Antigen constructs useful in the detection and differentiation of antibodies to HIV

ABSTRACT

Isolated HIV-1 Group O env polypeptides obtained from the HIV-1 isolate HAM112 are claimed, as well as (a) antigen constructs comprising fusions of one or more of each of HIV-1 Group O env polypeptides and HIV-1 Group M env polypeptide and (b) further antigen constructs containing additional Group O sequences and especially the gp41 IDR of isolate HAM112. Also claimed are polynucleotide sequences encoding the above, expression vectors comprising the same, host cells transformed thereby, and immunoassay methods and kits utilizing the antigen constructs of the invention.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. patent application Ser. No.11/008,351 filed on Dec. 9, 2004, which is a divisional of U.S. patentapplication Ser. No. 08/911,824 filed on Aug. 15, 1997, which is nowU.S. Pat. No. 6,846,905.

BACKGROUND OF THE INVENTION

This invention relates generally to immunoassays for the detection anddifferentiation of antibodies to Human Immunodeficiency Virus Type 1(HIV-1) Group M, HIV-1 Group O and Human Immunodeficiency Virus Type 2(HIV-2). More particularly, the invention relates to novel antigenconstructs useful as reagents in such assays, as well aspolynucleotides, DNA clones, expression vectors, transformed host cellsand the like which are useful in the preparation of such antigens.

Detection of HIV infection in a patient, and characterization of theviral type, are typically carried out using immunoassays which rely onthe highly specific interaction between antigens used as reagents in theassay and circulating antibodies in the patient's serum. Theimmunoreactivity of patient antibodies with some antigens, and to alesser extent or not at all with others, permits the identification ofthe type and subtype of the HIV which is present.

Currently, there are two major phylogenetic groups of HIV-1 designatedas Groups “M” and “O.” G. Meyers et al., Human Retroviruses and AIDS1995, Los Alamos National Laboratory, Los Alamos, N. Mex. (1995). HIV-1Group M isolates further have been divided into subgroups (A to J) thatare phylogenetically approximately equidistant from each other. Group Misolates predominate worldwide. The earliest reports about the sequenceof HIV-1 Group O indicated that these viruses were as closely related toa chimpanzee virus as to other HIV-1 subgroups. See, for example, L. G.Gürtler et al., J. Virology 68:1581-1585 (1994); M. Vanden Haesevelde etal., J. Virology 68:1586-1596 (1994); De Leys et al., J. Virology64:1207-1216 (1990); DeLeys et al., U.S. Pat. No. 5,304,466; L. G.Gürtler et al., European Patent Publication No. 591914 A2. The Group Osequences are the most divergent of the HIV-1 sequences described todate. Although HIV-1 Group O strains are endemic to west central Africa(Cameroon, Equatorial Guinea, Nigeria and Gabon), patients infected withGroup O isolates now have been identified in Belgium, France, Germany,Spain and the United States. See, for example, R. DeLeys et al., supra;P. Charneau et al., Virology 205:247-253 (1994); I. Loussert-Ajaka etal., J. Virology 69:5640-5649 (1995); H. Hampl et al., Infection23:369-370 (1995); A. Mas et al., AIDS Res. Hum. Retroviruses12:1647-1649 (1996); M. Peters et al., AIDS 11:493-498 (1997); and M. A.Rayfield et al., Emerging Infectious Diseases 2:209-212 (1996).

HIV-1 Group M serology is characterized in large part by the amino acidsequences of the expressed viral proteins (antigens), particularly thosecomprising the core and envelope (env) regions. As between variousstrains of this rapidly-mutating virus, these antigens are structurallyand functionally similar but have divergent amino acid sequences whichelicit antibodies that are similar but not identical in theirspecificity for a particular antigen.

One of the key serological targets for detection of HIV-1 infection isthe 41,000 MW transmembrane protein (TMP), glycoprotein 41 (gp41). gp41is a highly immunogenic protein which elicits a strong and sustainedantibody response in individuals considered seropositive for HIV.Antibodies to this protein are among the first to appear atseroconversion. The immune response to gp41 apparently remainsrelatively strong throughout the course of the disease, as evidenced bythe near universal presence of anti-gp41 antibodies in asymptomaticpatients as well as those exhibiting clinical stages of AIDS. Asignificant proportion of the antibody response to gp41 is directedtoward a well-characterized immunodominant region (IDR) within gp41.

Infections with HIV Type 2 (HIV-2), a virus initially found inindividuals from Africa, now have been identified in humans outside ofthe initial endemic area of West Africa, and have been reported inEuropeans who have lived in West Africa or those who have had sexualrelations with individuals from this region. See, for example, A. G.Saimot et al., Lancet i:688 (1987); M. A. Rey et al., Lancet i:388-389(1987); A. Werner et al., Lancet i:868-869 (1987); G. Brucker et al.,Lancet i:223 (1987); K. Marquart et al., AIDS 2:141 (1988); CDC, MMWR37:33-35 (1987); Anonymous, Nature 332:295 (1988). Cases of AIDS due toHIV-2 have been documented world-wide. Serologic studies indicate thatwhile HIV-1 and HIV-2 share multiple common epitopes in their coreantigens, the envelope glycoproteins of these two viruses are much lesscross-reactive. F. Clavel, AIDS 1:135-140 (1987). This limitedcross-reactivity of the envelope antigens is believed to explain whycurrently available serologic assays for HIV-1 may fail to react withcertain sera from individuals with antibody to HIV-2. F. Denis et al.,J. Clin. Micro. 26:1000-1004 (1988). Recently-issued U.S. Pat. No.5,055,391 maps the HIV-2 genome and provides assays to detect the virus.

These viral strains are, for the most part, readily identified andcharacterized using commercially-available diagnostic tests. However,concerns have arisen regarding the capability of currently-availableimmunoassays, designed for the detection of antibody to HIV-1 (Group M)and/or HIV-2, to detect the presence of antibody to HIV-1 Group O. I.Loussert-Ajaka et al., Lancet 343:1393-1394 (1994); C. A. Schable etal., Lancet 344:1333-1334 (1994); L. Gürtler et al., J. Virol. Methods51:177-184 (1995). Although, to date, few patients outside of westCentral Africa have been found to be infected with HIV-1 Group Oisolates, health officials fear the emergence of this subtype in othergeographic areas as well.

Consequently, there is a continued need for new antigens, suitable foruse in immunoassays, which alone or in conjunction with other antigenspermit the recognition of all HIV-1 (Group M and Group O) and HIV-2isolates and/or infections.

SUMMARY OF THE INVENTION

It has now been found that certain polypeptides or combinations of areparticularly useful in the detection of HIV-1 Group O and other HIVinfections. Consequently, in a first aspect of the present invention isdisclosed an isolated HIV-1 Group O env polypeptide having an amino acidsequence consisting essentially of the sequence of SEQ ID NO:61representing the full-length env region of the HIV-1 Group O isolateHAM112. Similarly disclosed is an isolated HIV-1 Group O env polypeptidecomprising an immunoreactive portion of the above full-lengthpolypeptide, as well as polynucleotides encoding such polypeptides.

In a second aspect of the present invention, an antigen construct isdisclosed which comprises a first HIV-1 Group O env polypeptide fused toa second HIV-1 Group O env polypeptide. Preferably, the firstpolypeptide of such an antigen construct is a gp120 polypeptide and thesecond polypeptide is a gp41 polypeptide, optionally with a portion ofthe hydrophobic region of the gp41 polypeptide being deleted so as tofacilitate expression when expressed as a recombinant product. Alsopreferred among the above antigen constructs are those in which at leastone of the first and second HIV-1 Group O env polypeptides is derivedfrom HIV-1 Group O isolate HAM112, as are those in which the firstpolypeptide comprises an immunoreactive portion of the gp120 protein ofHIV-1 Group O isolate HAM112.

In the above Group O env constructs, the first polypeptide may have anamino acid sequence which consists essentially of residues 1 through 520of the sequence of SEQ ID NO:61, or alternatively an immunoreactiveportion thereof. A shortened and preferred first polypeptide is onehaving an amino acid sequence consisting essentially of residues 476through 520 of the sequence of SEQ ID NO:61. Along with any of the abovepolypeptides, the second polypeptide used in the constructs of theinvention may be an immunoreactive portion of the gp41 protein of HIV-1Group O isolate HAM112, from which a portion of the hydrophobic regionof the gp41 protein of HIV-1 Group O isolate HAM112 is optionallyabsent. In particular, the deleted portion may be that part of gp41which has an amino acid sequence consisting essentially of residues 690through 715 of the sequence of SEQ ID NO:61.

The above second polypeptide will preferably have an amino acid sequenceconsisting essentially of residues 521 through 873 of the sequence ofSEQ ID NO:61 or a portion thereof. More preferably, the secondpolypeptide may have an amino acid sequence consisting essentially ofresidues 47 through 373 of the sequence of SEQ ID NO:52; still morepreferably, the amino acid sequence may consist essentially of residues47 through 245 of the sequence of SEQ ID NO:48; and even morepreferably, the amino acid sequence may consist essentially of residues47 through 215 of the sequence of SEQ ID NO:58. Representative of theGroup O env constructs of the invention are constructs pGO-8PL,pGO-8CKS, pGO-9PL, pGO-9CKS, pGO-11PL and pGO-11CKS, as well as anyderivatives, variants and analogs thereof.

In a further aspect of the present invention, there is disclosed anantigen construct comprising a fusion of at least one HIV-1 Group O envpolypeptide with at least one HIV-1 Group M env polypeptide, and morepreferably an antigen construct comprising a fusion of:

(a) a first HIV-1 Group O env polypeptide;

(b) a second HIV-1 Group O env polypeptide;

(c) a first HIV-1 Group M env polypeptide; and

(d) a second HIV-1 Group M env polypeptide.

The HIV-1 Group M polypeptides in the above constructs may be derivedfrom an HIV-1 isolate of Subtype B, and preferably at least one isderived from HIV-1 Group M isolate HXB2R. In any of these Group O/GroupM env constructs, at least one of the HIV-1 Group O sequences may bederived from HIV-1 Group O isolate HAM112.

More particularly, the first Group O env polypeptide and the first GroupM env polypeptide may both be gp120 polypeptides, while the second GroupO env polypeptide and the second Group M env polypeptide may both begp41 polypeptides. To enhance expression, a portion of the hydrophobicregion of at least one of the gp41 polypeptides may be deleted. Antigenconstructs included among the above are those in which:

(a) the first HIV-1 Group O env polypeptide comprises an immunoreactiveportion of the gp120 protein of HIV-1 Group O isolate HAM112;

(b) the second HIV-1 Group O env polypeptide comprises an immunoreactiveportion of the gp41 protein of HIV-1 Group O isolate HAM112

(c) the first HIV-1 Group M env polypeptide comprises an immunoreactiveportion of the gp120 protein of a first HIV-1 Group M isolate of SubtypeB; and

(d) the second HIV-1 Group M env polypeptide comprises an immunoreactiveportion of the gp41 protein of a second HIV-1 Group M isolate of SubtypeB.

Preferred among these are constructs wherein the first and second HIV-1Group M isolates of Subtype B are the same and are HIV-1 Group M isolateHXB2R, as well as those wherein a portion of the hydrophobic region ofthe gp41 protein of HIV-1 Group M isolate HXB2R is absent from thesecond HIV-1 Group M env polypeptide.

Preferred Group O/Group M env constructs include those in which (a) thefirst HIV-1 Group M env polypeptide has an amino acid sequenceconsisting essentially of residues 251 through 292 of the sequence ofSEQ ID NO:108, and (b) the second HIV-1 Group M env polypeptide has anamino acid sequence consisting essentially of residues 293 through 599of the sequence of SEQ ID NO: 108 or a portion thereof. Especiallypreferred are those in which the second HIV-1 Group M env polypeptidehas an amino acid sequence consisting essentially of residues 293through 492 of the sequence of SEQ ID NO: 108.

Also preferred are the above Group O/Group M env constructs in which thefirst HIV-1 Group O env polypeptide has an amino acid sequenceconsisting essentially of residues 1 through 520 of the sequence of SEQID NO:61 or a portion thereof, and especially those comprising a firstHIV-1 Group O env polypeptide which has an amino acid sequenceconsisting essentially of residues 476 through 520 of the sequence ofSEQ ID NO:61. The second HIV-1 Group O env polypeptide may be one havingan amino acid sequence consisting essentially of residues 521 through873 of the sequence of SEQ ID NO:61 or a portion thereof, from which aportion of the hydrophobic region of the gp41 protein of HIV-1 Group βisolate HAM112 may optionally be absent. Preferred constructs are thosein which such second HIV-1 Group O env polypeptides have an amino acidsequence consisting essentially of residues 47 through 373 of thesequence of SEQ ID NO:52; more preferred are those in which the secondHIV-1 Group O env polypeptide has an amino acid sequence consistingessentially of residues 47 through 245 of the sequence of SEQ ID NO:48;and even more preferred are those in which the second HIV-1 Group O envpolypeptide has an amino acid sequence consisting essentially ofresidues 47 through 215 of the sequence of SEQ ID NO:58. Representativeof the Group O/Group M env constructs of the invention are constructspGO-12CKS, pGO-13CKS and pGO-14PL, and derivatives, variants and analogsthereof.

In yet another aspect of the present invention, an antigen construct isdisclosed which comprises a fusion of a first HIV-1 env polypeptide, asecond HIV-1 env polypeptide, and at least one additional HIV-1polypeptide, and especially one in which each such HIV-1 envpolypeptides are HIV-1 Group O polypeptides. The first HIV-1 Group O envpolypeptide of this construct may be a gp120 polypeptide, and the secondHIV-1 Group O env polypeptide a gp41 polypeptide. More particularly, thefirst HIV-1 Group O env polypeptide of this construct may comprise animmunoreactive portion of the gp120 protein of HIV-1 Group O isolateHAM112, while the second HIV-1 Group O env polypeptide may comprise animmunoreactive portion of the gp41 protein of HIV-1 Group O isolateHAM112.

Among these constructs, those in which the first HIV-1 Group O envpolypeptide has an amino acid sequence consisting essentially ofresidues 1 through 520 of the sequence of SEQ ID NO:61, or a portionthereof, are preferred; more preferred are those in which the firstHIV-1 Group O env polypeptide has an amino acid sequence consistingessentially of residues 476 through 520 of the sequence of SEQ ID NO:61.As to the second HIV-1 Group O env polypeptide, which may have an aminoacid sequence consisting essentially of residues 521 through 873 of thesequence of SEQ ID NO:61 or a portion thereof and from which a portionof the hydrophobic region of the gp41 protein of HIV-1 Group O isolateHAM112 may optionally be absent, preferred are those constructs in whichthat second HIV-1 Group O env polypeptide has an amino acid sequenceconsisting essentially of residues 47 through 373 of the sequence of SEQID NO:52. Even more preferred are those having a second HIV-1 Group Oenv polypeptide with an amino acid sequence consisting essentially ofresidues 47 through 245 of the sequence of SEQ ID NO:48, and especiallythose in which the amino acid sequence consists essentially of residues47 through 215 of the sequence of SEQ ID NO:58.

The additional HIV-1 polypeptide in any of these constructs may be aGroup O env polypeptide; however, it is intended that it mayalternatively be an immunogenic polypeptide from any of HIV-1 Groups Mor O or HIV-2, including env, gag, pol, reverse transcriptase, andregulatory and other viral components. Preferred in any case are thoseconstructs in which the additional HIV-1 Group O polypeptide comprisesan immunoreactive portion of the gp41 protein of HIV-1 Group O isolateHAM112. Also preferred are those wherein the additional HIV-1 Group Opolypeptide has an amino acid sequence consisting essentially ofresidues 521 through 873 of the sequence of SEQ ID NO:61 or a portionthereof, from which the hydrophobic region of the gp41 protein of HIV-1Group O isolate HAM112 may optionally be absent. Even more preferred areconstructs in which the additional HIV-1 Group O env polypeptide has anamino acid sequence consisting essentially of residues 47 through 373 ofthe sequence of SEQ ID NO:52; particularly preferred are those in whichthe additional HIV-1 Group O env polypeptide has an amino acid sequenceconsisting essentially of residues 47 through 245 of the sequence of SEQID NO:48, and especially those wherein the additional HIV-1 Group O envpolypeptide has an amino acid sequence consisting essentially ofresidues 47 through 215 of the sequence of SEQ ID NO:58. Most preferredare constructs having as the additional HIV-1 Group O env polypeptidethe so-called immunodominant region (IDR) of HIV-1 Group O, which has anamino acid sequence consisting essentially of residues 250 through 281of the sequence of SEQ ID NO:120. Representative of the above constructsare pGO-15CKS and pGO-15PL, as well as any derivatives, variants andanalogs thereof.

In still another aspect of the present invention is disclosed an antigenconstruct comprising a first HIV-2 env polypeptide fused to a secondHIV-2 env polypeptide, and especially one in which the first HIV-2 envpolypeptide is a gp120 polypeptide and the second HIV-2 env polypeptideis a gp36 polypeptide. Preferred among the such constructs are those inwhich:

(a) the first HIV-2 env polypeptide has an amino acid sequenceconsisting essentially of residues 248 through 307 of the sequence ofSEQ ID NO:55 or a portion thereof; and

(b) the second HIV-2 env polypeptide has an amino acid sequenceconsisting essentially of residues 308 through 466 of the sequence ofSEQ ID NO:55 or a portion thereof.

Representative of the HIV-2 constructs of the invention is pHIV-210 (SEQID NO:55), as well as any derivatives, variants and analogs thereof.

An additional aspect of the present invention comprises polynucleotidesencoding any of the above antigen construct, which polynucleotide may beoperably linked to a control sequence capable of directing expression ina suitable host and/or have a coding sequence which has been modified toprovide a codon bias appropriate to the expression host. Still otheraspects of the present invention include expression vectors comprisingsuch polynucleotides and host cells transformed thereby, particularlywhere the host is Escherichia coli.

In a further aspect of the present invention, there is disclosed amethod for detecting antibodies to HIV-1 in a test sample comprising thesteps of:

(a) combining at least one antigen construct according to the inventionwith the test sample to form a mixture;

(b) incubating the mixture under conditions suitable for formation ofcomplexes between the antigen and antibodies, if any, which are presentin the sample and are immunologically reactive with the antigen; and

(c) detecting the presence of any complexes formed.

In one embodiment of the method, detection of the presence of complexesin step (c) is carried out using an additional antigen construct of theinvention to which a signal-generating compound has been attached. Inanother embodiment, detection is carried out using an additional antigenconstruct of the invention to which a first member of a specific bindingpair is attached, and further using an indicator reagent comprising asecond member of the specific binding pair to which is attached asignal-generating compound. A further embodiment provides that detectionof the presence of complexes in step (c) is carried out using anantibody directed to the complexes formed in step (b), to which antibodyis attached a signal-generating compound. Still another embodimentprovides that detection of the presence of complexes in step (c) iscarried out using an antibody directed to the complexes formed in step(b) and attached thereto a first member of a specific binding pair; suchdetection further requires the use of an indicator reagent comprising asecond member of the specific binding pair to which is attached asignal-generating compound.

In a final aspect of the present invention are disclosed immunoassaykits for the detection of antibodies to HIV-1, which kits comprise anantigen construct of the invention. Such construct may be used as acapture reagent or an indicator reagent. Alternatively, the antigenconstruct may be attached to a first member of a specific binding pair,the kit additionally comprising an indicator reagent comprising a secondmember of the specific binding pair attached to a signal-generatingcompound.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of an isolated polypeptide of the present invention,the amino acid sequence of the env protein of the HIV-1 Group O isolateHAM112 is shown as SEQ ID NO:61. In the present context, “isolated” isintended to mean that such polypeptides are relatively purified withrespect to other viral or cellular components which normally would bepresent in situ, up to and including a substantially pure preparation ofthe protein. Such polypeptides can be utilized as assay reagents, forthe production of monoclonal or polyclonal antibodies, in themanufacture of vaccines, or otherwise.

Immunoreactive portions, or fragments, of the above polypeptides arealso expected to be useful. By “immunoreactive” is meant portions ofsuch length as are capable of eliciting an immune response in a hostand/or of reacting with antibodies directed specifically thereto;preferably, such partial polypeptides will be five or more amino acidsin length. It should also be noted that the term “portion” as usedherein is directed to both terminally truncated sequences and thosewhich are shortened by the removal of an intervening sequence.

The above polypeptides and portions will best be produced by expressionof polynucleotides encoding the same. These too permit a degree ofvariability in their sequence, as for example due to degeneracy of thegenetic code, codon bias in favor of the host cell expressing thepolypeptide, and conservative amino acid substitutions in the resultingprotein. Moreover, it is anticipated that some variation of sequenceswill occur between—and possibly even within—a given HIV-1 isolate orother phylogenetic unit. Consequently, the polypeptides and constructsof the invention include not only those which are identical in sequenceto the above sequence but also those which have an amino acid sequencethat consist essentially of that reference sequence, where the term“consisting essentially” is meant to embrace variant polypeptides thestructural and functional characteristics of which remain substantiallythe same. Preferably, such variants (or “analogs”) will have a sequencehomology (“identity”) of 80% or more with the reference sequence of SEQID NO:61. In this sense, techniques for determining amino acid sequence“similarity” are well-known in the art. In general, “similarity” meansthe exact amino acid to amino acid comparison of two or morepolypeptides at the appropriate place, where amino acids are identicalor possess similar chemical and/or physical properties such as charge orhydrophobicity. A so-termed “percent similarity” then can be determinedbetween the compared polypeptide sequences. Techniques for determiningnucleic acid and amino acid sequence identity also are well known in theart and include determining the nucleotide sequence of the mRNA for thatgene (usually via a cDNA intermediate) and determining the amino acidsequence encoded therein, and comparing this to a second amino acidsequence. In general, “identity” refers to an exact nucleotide tonucleotide or amino acid to amino acid correspondence of twopolynucleotides or polypeptide sequences, respectively. Two or morepolynucleotide sequences can be compared by determining their “percentidentity”, as can two or more amino acid sequences. The programsavailable in the Wisconsin Sequence Analysis Package, Version 8(available from Genetics Computer Group, Madison, Wis.), for example,the GAP program, are capable of calculating both the identity betweentwo polynucleotides and the identity and similarity between twopolypeptide sequences, respectively. Other programs for calculatingidentity or similarity between sequences are known in the art.

According to another embodiment of the invention, antigen constructs areprovided which are suitable for use in the detection of anti-HIV-1antibodies. As described in greater detail below, such constructs may beprepared by recombinant means, as synthetic peptides, or otherwise;moreover, they may be glycosylated or unglycosylated depending on themanner and/or host cell by which they are made. Consequently, althoughreferred to as if comprising glycoproteins (for example, “a gp120polypeptide”), the antigen constructs of the invention are intended toinclude those which are expressed in bacterial hosts such as E. coli andare therefore unglycosylated.

It should be noted that the above constructs are fusions of varioussequences, that is, the constructs are formed by joining variousepitope-containing sequences, as for example by co-expression, ligationor sequential synthesis. Also joined thereto, and optionally included inthe constructs of the invention, are other polypeptide sequences such asexpression (CKS) polylinkers and other linker sequences. The order ofthe various polypeptide sequences is not critical; consequently, thepolypeptides and their epitopes may be re-arranged as a matter ofconvenience. Further modifications are also possible, as for example byrandom mutation or site-directed mutagenesis or even the deletion(removal or omission) of certain regions such as the gp41 hydrophobicregion, the absence of which has been found to enhance expression of theremaining polypeptide. In any case, whether nearly the same orsubstantially changed, polypeptides which undergo these modificationsmay be said to be “derived” from their respective sources, and theresulting polypeptides may be regarded as “derivatives”.

In yet another aspect of the present invention, assay methods areprovided which utilize the constructs of the invention in the detectionof anti-HIV-1 antibodies in test samples. Such methods permit the directtesting of biological specimens; however, the assay methods may also bemodified to permit the testing of pre-processed specimens such as sera,lysed cells, and extracts or preparations made therefrom (as byconcentration, dilution, separation, fixation and/or immobilization).Depending on the desired assay format, the antigen constructs may alsobe modified for use in such assays, as for example by labeling,immobilization on a solid phase or otherwise, or conjugation to otherassay reagents.

Certain terms used herein are intended to have specialized meanings.Unless otherwise stated, the terms below shall have the followingmeanings:

The term “primer” denotes a specific oligonucleotide sequencecomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. It serves as an initiation point fornucleotide polymerization catalyzed by either DNA polymerase, RNApolymerase or reverse transcriptase.

The term “polynucleotide” as used herein means a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes double- and single-stranded DNA aswell as double- and single-stranded RNA. It also includes modifications,such as methylation or capping, and unmodified forms of thepolynucleotide.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence. Also encompassed are polypeptide sequences whichare immunologically identifiable with a polypeptide encoded by thesequence. Thus, a “polypeptide,” “protein,” or “amino acid” sequence asclaimed herein may have at least 60% similarity, more preferably atleast about 70% similarity, and most preferably about 80% similarity toa particular polypeptide or amino acid sequence specified below.

The terms “recombinant polypeptide” or “recombinant protein”, usedinterchangeably herein, describe a polypeptide which by virtue of itsorigin or manipulation is not associated with all or a portion of thepolypeptide with which it is associated in nature and/or is linked to apolypeptide other than that to which it is linked in nature. Arecombinant or encoded polypeptide or protein is not necessarilytranslated from a designated nucleic acid sequence. It also may begenerated in any manner, including chemical synthesis or expression of arecombinant expression system.

“Polypeptide” and “protein” are used interchangeably herein and indicatea molecular chain of amino acids linked through covalent and/ornoncovalent bonds. The terms do not refer to a specific length of theproduct. Thus, peptides, oligopeptides and proteins are included withinthe definition of polypeptide. The terms include post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. In addition, proteinfragments, analogs, mutated or variant proteins, fusion proteins and thelike are included within the meaning of polypeptide.

A “fragment” of a specified polypeptide refers to an amino acid sequencewhich comprises at least about 3-5 amino acids, more preferably at leastabout 8-10 amino acids, and even more preferably at least about 15-20amino acids, derived from the specified polypeptide.

The term “synthetic peptide” as used herein means a polymeric form ofamino acids of any length, which may be chemically synthesized bymethods well-known to those skilled in the art. These synthetic peptidesare useful in various applications.

“Purified polypeptide” means a polypeptide of interest or fragmentthereof which is essentially free, that is, contains less than about50%, preferably less than about 70%, and more preferably, less thanabout 90% of cellular components with which the polypeptide of interestis naturally associated. Methods for purifying are known in the art.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, which is separated from some orall of the coexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

“Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cellcultures,” and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vector orother transferred DNA, and include the original progeny of the originalcell which has been transfected.

As used herein “replicon” means any genetic element, such as a plasmid,a chromosome or a virus, that behaves as an autonomous unit ofpolynucleotide replication within a cell.

A “vector” is a replicon to which another polynucleotide segment isattached, such as to bring about the replication and/or expression ofthe attached segment.

The term “control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, such control sequences generally includepromoter, ribosomal binding site and terminators; in eukaryotes, suchcontrol sequences generally include promoters, terminators and, in someinstances, enhancers. The term “control sequence” thus is intended toinclude at a minimum all components whose presence is necessary forexpression, and also may include additional components whose presence isadvantageous, for example, leader sequences.

“Operably linked” refers to a situation wherein the components describedare in a relationship permitting them to function in their intendedmanner. Thus, for example, a control sequence “operably linked” to acoding sequence is ligated in such a manner that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

A “coding sequence” is a polynucleotide sequence which is transcribedinto mRNA and translated into a polypeptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by and include a translation start codonat the 5′-terminus and one or more translation stop codons at the3′-terminus. A coding sequence can include, but is not limited to, mRNA,cDNA, and recombinant polynucleotide sequences.

The term “immunologically identifiable with/as” refers to the presenceof epitope(s) and polypeptide(s) which also are present in and areunique to the designated polypeptide(s). Immunological identity may bedetermined by antibody binding and/or competition in binding. Thesetechniques are known to the skilled artisan and also are describedherein. The uniqueness of an epitope also can be determined by computersearches of known data banks, such as GenBank, for the polynucleotidesequences which encode the epitope, and by amino acid sequencecomparisons with other known proteins.

As used herein, “epitope” means an antigenic determinant of apolypeptide. Conceivably, an epitope can comprise three amino acids in aspatial conformation which is unique to the epitope. Generally, anepitope consists of at least five such amino acids, and more usually, itconsists of at least eight to ten amino acids. Methods of examiningspatial conformation are known in the art and include, for example,x-ray crystallography and two-dimensional nuclear magnetic resonance.

A “conformational epitope” is an epitope that is comprised of specificjuxtaposition of amino acids in an immunologically recognizablestructure, such amino acids being present on the same polypeptide in acontiguous or non-contiguous order or present on different polypeptides.

A polypeptide is “immunologically reactive” with an antibody when itbinds to an antibody due to antibody recognition of a specific epitopecontained within the polypeptide. Immunological reactivity may bedetermined by antibody binding, more particularly by the kinetics ofantibody binding, and/or by competition in binding using ascompetitor(s) a known polypeptide(s) containing an epitope against whichthe antibody is directed. The methods for determining whether apolypeptide is immunologically reactive with an antibody are known inthe art.

The term “transformation” refers to the insertion of an exogenouspolynucleotide into a host cell, irrespective of the method used for theinsertion. For example, direct uptake, transduction or f-mating areincluded. The exogenous polynucleotide may be maintained as anon-integrated vector, for example, a plasmid, or alternatively, may beintegrated into the host genome.

The term “test sample” refers to a component of an individual's bodywhich is the source of the analyte (such as, antibodies of interest orantigens of interest). These components are well known in the art. Thesetest samples include biological samples which can be tested by themethods of the present invention described herein and include human andanimal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, lymph fluids, and various external secretions of therespiratory, intestinal and genitorurinary tracts, tears, saliva, milk,white blood cells, myelomas and the like; biological fluids such as cellculture supernatants; fixed tissue specimens; and fixed cell specimens.

“Purified product” refers to a preparation of the product which has beenisolated from the cellular constituents with which the product isnormally associated, and from other types of cells which may be presentin the sample of interest.

The present invention provides assays which utilize specific bindingmembers. A “specific binding member,” as used herein, is a member of aspecific binding pair. That is, two different molecules where one of themolecules through chemical or physical means specifically binds to thesecond molecule. Therefore, in addition to antigen and antibody specificbinding pairs of common immunoassays, other specific binding pairs caninclude biotin and avidin, carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors and enzymes, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, antibodies and antibody fragments, both monoclonal andpolyclonal, and complexes thereof, including those formed by recombinantDNA molecules.

A “capture reagent,” as used herein, refers to an unlabeled specificbinding member which is specific either for the analyte as in a sandwichassay, for the indicator reagent or analyte as in a competitive assay,or for an ancillary specific binding member, which itself is specificfor the analyte, as in an indirect assay. The capture reagent can bedirectly or indirectly bound to a solid phase material before theperformance of the assay or during the performance of the assay, therebyenabling the separation of immobilized complexes from the test sample.

The “indicator reagent” comprises a “signal-generating compound”(“label”) which is capable of generating and generates a measurablesignal detectable by external means, conjugated (“attached”) to aspecific binding member. “Specific binding member” as used herein meansa member of a specific binding pair. That is, two different moleculeswhere one of the molecules through chemical or physical meansspecifically binds to the second molecule. In addition to being anantibody member of a specific binding pair, the indicator reagent alsocan be a member of any specific binding pair, including eitherhapten-anti-hapten systems such as biotin or anti-biotin, avidin orbiotin, a carbohydrate or a lectin, a complementary nucleotide sequence,an effector or a receptor molecule, an enzyme cofactor and an enzyme, anenzyme inhibitor or an enzyme, and the like. An immunoreactive specificbinding member can be an antibody, an antigen, or an antibody/antigencomplex that is capable of binding either to polypeptide of interest asin a sandwich assay, to the capture reagent as in a competitive assay,or to the ancillary specific binding member as in an indirect assay.

The various “signal-generating compounds” (labels) contemplated includechromogens, catalysts such as enzymes, luminescent compounds such asfluorescein and rhodamine, chemiluminescent compounds such asdioxetanes, acridiniums, phenanthridiniums and luminol, radioactiveelements, and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase, and the like.The selection of a particular label is not critical, but it will becapable of producing a signal either by itself or in conjunction withone or more additional substances.

“Solid phases” (“solid supports”) are known to those in the art andinclude the walls of wells of a reaction tray, test tubes, polystyrenebeads, magnetic beads, nitrocellulose strips, membranes, microparticlessuch as latex particles, sheep (or other animal) red blood cells, andDuracytes® (red blood cells “fixed” by pyruvic aldehyde andformaldehyde, available from Abbott Laboratories, Abbott Park, Ill.) andothers. The “solid phase” is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and Duracytes® are all suitable examples. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like. A “solid phase”, as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid phase can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid phase and which has the ability to immobilize the capturereagent through a specific binding reaction. The receptor moleculeenables the indirect binding of the capture reagent to a solid phasematerial before the performance of the assay or during the performanceof the assay. The solid phase thus can be a plastic, derivatizedplastic, magnetic or non-magnetic metal, glass or silicon surface of atest tube, microtiter well, sheet, bead, microparticle, chip, sheep (orother suitable animal's) red blood cells, Duracytes® and otherconfigurations known to those of ordinary skill in the art.

It is contemplated and within the scope of the present invention thatthe solid phase also can comprise any suitable porous material withsufficient porosity to allow access by detection antibodies and asuitable surface affinity to bind antigens. Microporous structuregenerally are preferred, but materials with gel structure in thehydrated state may be used as well. Such useful solid supports includebut are not limited to nitrocellulose and nylon. It is contemplated thatsuch porous solid supports described herein preferably are in the formof sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1mm. The pore size may vary within wide limits, and preferably is fromabout 0.025 to 15 microns, especially from about 0.15 to 15 microns. Thesurface of such supports may be activated by chemical processes whichcause covalent linkage of the antigen or antibody to the support. Theirreversible binding of the antigen or antibody is obtained, however, ingeneral, by adsorption on the porous material by poorly understoodhydrophobic forces. Other suitable solid supports are known in the art.

The present invention provides polynucleotide sequences derived fromhuman immunodeficiency viruses of interest and polypeptides encodedthereby. The polynucleotide(s) may be in the form of mRNA or DNA.Polynucleotides in the form of DNA, cDNA, genomic DNA, and synthetic DNAare within the scope of the present invention. The DNA may bedouble-stranded or single-stranded, and if single stranded may be thecoding (sense) strand or non-coding (anti-sense) strand. The codingsequence which encodes the polypeptide may be identical to the codingsequence provided herein or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same polypeptide as the DNA provided herein.

This polynucleotide may include only the coding sequence for thepolypeptide, or the coding sequence for the polypeptide and additionalcoding sequence such as a leader or secretory sequence or a proproteinsequence, or the coding sequence for the polypeptide (and optionallyadditional coding sequence) and non-coding sequence, such as anon-coding sequence 5′ and/or 3′ of the coding sequence for thepolypeptide.

In addition, the invention includes variant polynucleotides containingmodifications such as polynucleotide deletions, substitutions oradditions; and any polypeptide modification resulting from the variantpolynucleotide sequence. A polynucleotide of the present invention alsomay have a coding sequence which is a naturally-occurring variant of thecoding sequence provided herein.

In addition, the coding sequence for the polypeptide may be fused in thesame reading frame to a polynucleotide sequence which aids in expressionand secretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the proteinplus additional 5′ amino acid residues. A protein having a prosequenceis a proprotein and may in some cases be an inactive form of theprotein. Once the prosequence is cleaved an active protein remains.Thus, the polynucleotide of the present invention may encode for aprotein, or for a protein having a prosequence or for a protein havingboth a presequence (leader sequence) and a prosequence.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the polypeptide fused to the marker in thecase of a bacterial host, or, for example, the marker sequence may be ahemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein. See, for example, I. Wilson et al., Cell 37:767(1984).

The present invention further relates to HIV-1 polypeptides which havethe deduced amino acid sequence as provided herein, as well asfragments, analogs and derivatives of such polypeptides. Thepolypeptides of the present invention may be recombinant polypeptides,natural purified polypeptides or synthetic polypeptides. The fragment,derivative or analog of such a polypeptide may be one in which one ormore of the amino acid residues is substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or it may be one in which one or more ofthe amino acid residues includes a substituent group; or it may be onein which the polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol); or it may be one in which the additional aminoacids are fused to the polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of thepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are within the scope of the present invention. The polypeptidesand polynucleotides of the present invention are preferably provided inan isolated form, and preferably purified.

Thus, a polypeptide of the present invention may have an amino acidsequence that is identical to that of the naturally-occurringpolypeptide or that is different by minor variations due to one or moreamino acid substitutions. The variation may be a “conservative change”typically in the range of about 1 to 5 amino acids, wherein thesubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine or threonine with serine.In contrast, variations may include nonconservative changes, e.g.,replacement of a glycine with a tryptophan. Similar minor variations mayalso include amino acid deletions or insertions, or both. Guidance indetermining which and how many amino acid residues may be substituted,inserted or deleted without changing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software (DNASTAR Inc., Madison Wis.).

The recombinant polypeptides of the present invention can be producednot only as demonstrated below, but also according to a number ofalternative methods and using a variety of host cells and expressionvectors. Host cells are genetically engineered (transduced ortransformed or transfected) with the vectors of this invention which maybe a cloning vector or an expression vector. The vector may be in theform of a plasmid, a viral particle, a phage, etc. The engineered hostcells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying HIV-derived genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The polynucleotides of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, thepolynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,and pseudorabies. However, any other plasmid or vector may be used solong as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted intoappropriate restriction endonuclease sites by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art. The DNA sequence in the expression vector isoperatively linked to an appropriate expression control sequence(s)(promoter) to direct mRNA synthesis. Representative examples of suchpromoters include but are not limited to LTR or SV40 promoter, the E.coli lac or trp, the phage lambda P sub L promoter and other promotersknown to control expression of genes in prokaryotic or eukaryotic cellsor their viruses. The expression vector also contains a ribosome bindingsite for translation initiation and a transcription terminator. Thevector may also include appropriate sequences for amplifying expression.In addition, the expression vectors preferably contain a gene to providea phenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium; Streptomyces sp.; fungal cells, such as yeast; insect cellssuch as Drosophila and Sf9; animal cells such as chinese hamster ovary(CHO), COS or Bowes melanoma; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings provided herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pINCY (Incyte PharmaceuticalsInc., Palo Alto, Calif.), pSPORT1 (Life Technologies, Gaithersburg,Md.), pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript, psiX174,pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic:pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL(Pharmacia). However, any other plasmid or vector may be used as long asit is replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, SP6, T7, gpt, lambda P subR, P sub L and trp. Eukaryotic promoters include cytomegalovirus (CMV)immediate early, herpes simplex virus (HSV) thymidine kinase, early andlate SV40, LTRs from retrovirus, and mouse metallothionein-I. Selectionof the appropriate vector and promoter is well within the level ofordinary skill in the art.

The host cell used herein can be a higher eukaryotic cell, such as amammalian cell, or a lower eukaryotic cell, such as a yeast cell, or thehost cell can be a prokaryotic cell, such as a bacterial cell.Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-Dextran mediated transfection, orelectroporation (L. Davis et al., “Basic Methods in Molecular Biology”,2nd edition, Appleton and Lang, Paramount Publishing, East Norwalk,Conn. [1994]).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems also can be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, (Cold Spring Harbor, N.Y., 1989), which is herebyincorporated by reference.

Transcription of a DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication origin,and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and the S.cerevisiae TRP1 gene, and a promoter derived from a highly-expressedgene to direct transcription of a downstream structural sequence. Suchpromoters can be derived from operons encoding glycolytic enzymes suchas 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, orheat shock proteins, among others. The heterologous structural sequenceis assembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a routine matter of choice.

Useful expression vectors for bacterial use comprise a selectable markerand bacterial origin of replication derived from plasmids comprisinggenetic elements of the well-known cloning vector pBR322 (ATCC 37017).Other vectors include but are not limited to PKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.).These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction), and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents; suchmethods are well-known to the ordinary artisan.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, such as the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalianexpression vectors will comprise an origin of replication, a suitablepromoter and enhancer, and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, 5′ flanking nontranscribed sequences, andselectable markers such as the neomycin phosphotransferase gene. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements. Representative,useful vectors include pRc/CMV and pcDNA3 (available from Invitrogen,San Diego, Calif.).

The HIV-derived polypeptides are recovered and purified from recombinantcell cultures by known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxyapatite chromatography or lectin chromatography. It is preferredto have low concentrations (approximately 0.1-5 mM) of calcium ionpresent during purification (Price et al., J. Biol. Chem. 244:917[1969]). Protein refolding steps can be used, as necessary, incompleting configuration of the protein. Finally, high performanceliquid chromatography (HPLC) can be employed for final purificationsteps.

The polypeptides of the present invention may be naturally purifiedproducts expressed from a high expressing cell line, or a product ofchemical synthetic procedures, or produced by recombinant techniquesfrom a prokaryotic or eukaryotic host (for example, by bacterial, yeast,higher plant, insect and mammalian cells in culture). Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated with mammalian or othereukaryotic carbohydrates or may be non-glycosylated. The polypeptides ofthe invention may also include an initial methionine amino acid residue.

The present invention further includes modified versions of therecombinant polypeptide to preclude glycosylation while allowingexpression of a reduced carbohydrate form of the protein in yeast,insect or mammalian expression systems. Known methods for inactivatingglycosylation sites include, but are not limited to, those presented inU.S. Pat. No. 5,071,972 and EP 276,846, which are incorporated herein byreference.

Other variants included in the present invention include those obtainedby removal removal of sequences encoding cystein residues, therebypreventing formation of incorrect intramolecular disulfide bridges whichdecrease biological activity of the protein product. The constructs ofthe present invention also may be prepared by removal of the site ofproteolytic processing, allowing expression in systems which contain aproblematic protease, for example the KEX2 protease in yeast. Knownmethods for removing such protease sites include but are not limited toone method for removing KEX2 sites presented in EP212,914.

The present invention includes the above peptides in the form ofoligomers, dimers, trimers and higher order oligomers. Oligomers may beformed by several means including but not limited to disulfide bondsbetween peptides, non-covalent interactions between peptides, andpoly-ethylene-glycol linkages between peptides.

The fusion of the above peptides to peptide linkers or peptides that arecapable of promoting oligomers is also encompassed in this invention.Such peptides include but are not limited to leucine zippers andantibody derived peptides, such as is described in Landschulz et al.,Science 240:1759 (1988); Hollenbaugh and Aruffo, “Construction ofImmunoglobin Fusion Proteins”, in Current Protocols in Immunology,Supplement 4, pgs 10.19.1-10.19.11 (1992) John Wiley and sons, New York,N.Y.

The starting plasmids can be constructed from available plasmids inaccord with published, known procedures. In addition, equivalentplasmids to those described are known in the art and will be apparent tothe ordinarily skilled artisan.

Once homogeneous cultures of recombinant cells are obtained, largequantities of recombinantly produced protein can be recovered from theconditioned medium and analyzed using chromatographic methods well knownin the art. An alternative method for the production of large amounts ofsecreted protein involves the transformation of mammalian embryos andthe recovery of the recombinant protein from milk produced by transgeniccows, goats, sheep, etc. Polypeptides and closely related molecules maybe expressed recombinantly in such a way as to facilitate proteinpurification. One approach involves expression of a chimeric proteinwhich includes one or more additional polypeptide domains not naturallypresent on human polypeptides. Such purification-facilitating domainsinclude, but are not limited to, metal-chelating peptides such ashistidine-tryptophan domains that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle, Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase fromInvitrogen (San Diego, Calif.) between the polypeptide sequence and thepurification domain may be useful for recovering the polypeptide.

It is also contemplated and within the scope of the present inventionthat the above recombinant antigens will be used in a variety ofimmunoassay formats, including but not limited to direct and indirectassays. The means for adapting the antigens to such various formats—asby conjugation to labels or macromolecules, or immobilization onsuitable support surfaces—are well-understood and should be familiar tothose skilled in the art.

For example, the polypeptides including their fragments or derivativesor analogs thereof of the present invention, or cells expressing them,can be used for the detection of antibodies to HIV (as well as animmunogen to produce antibodies). These antibodies can be, for example,polyclonal or monoclonal antibodies, chimeric, single chain andhumanized antibodies, as well as Fab fragments, or the product of an Fabexpression library. Various procedures known in the art may be used forthe production of such antibodies and fragments.

Further, antibodies generated against a polypeptide corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptide into an animal or by administering the polypeptide to ananimal such as a mouse, rabbit, goat or human. A mouse, rabbit or goatis preferred. The antibody so obtained then will bind the polypeptideitself. In this manner, even a sequence encoding only a fragment of thepolypeptide can be used to generate antibodies that bind the nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom test samples such as tissue suspected of containing thatpolypeptide. For preparation of monoclonal antibodies, any techniquewhich provides antibodies produced by continuous cell line cultures canbe used. Examples include the hybridoma technique as described by Kohlerand Milstein, Nature 256:495-497 (1975), the trioma technique, the humanB-cell hybridoma technique as described by Kozbor et al, Immun. Today4:72 (1983), and the EBV-hybridoma technique to produce human monoclonalantibodies as described by Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc, New York, N.Y., pp. 77-96 (1985).Techniques described for the production of single chain antibodies canbe adapted to produce single chain antibodies to immunogenic polypeptideproducts of this invention. See, for example, U.S. Pat. No. 4,946,778,which is incorporated herein by reference.

Various assay formats may utilize such antibodies, including “sandwich”immunoassays and probe assays. For example, the monoclonal antibodies orfragment as described above can be employed in various assay systems todetermine the presence, if any, of HIV-derived polypeptide in a testsample. For example, in a first assay format, a polyclonal or monoclonalantibody or fragment thereof, or a combination of these antibodies,which has been coated on a solid phase, is contacted with a test sample,to form a first mixture. This first mixture is incubated for a time andunder conditions sufficient to form antigen/antibody complexes. Then, anindicator reagent comprising a monoclonal or a polyclonal antibody or afragment thereof, or a combination of these antibodies, to which asignal generating compound has been attached, is contacted with theantigen/antibody complexes to form a second mixture. This second mixturethen is incubated for a time and under conditions sufficient to formantibody/antigen/antibody complexes. The presence of an HIV-derivedpolypeptide antigen present in the test sample and captured on the solidphase, if any, is determined by detecting the measurable signalgenerated by the signal generating compound. The amount of HIV-derivedpolypeptide antigen present in the test sample is proportional to thesignal generated.

Or, a polyclonal or monoclonal HIV-derived polypeptide antibody orfragment thereof, or a combination of these antibodies which is bound toa solid support, the test sample and an indicator reagent comprising amonoclonal or polyclonal antibody or fragments thereof, whichspecifically binds to HIV-derived polypeptide antigen, or a combinationof these antibodies to which a signal generating compound is attached,are contacted to form a mixture. This mixture is incubated for a timeand under conditions sufficient to form antibody/antigen/antibodycomplexes. The presence, if any, of HIV-derived polypeptide present inthe test sample and captured on the solid phase is determined bydetecting the measurable signal generated by the signal generatingcompound. The amount of HIV-derived polypeptide proteins present in thetest sample is proportional to the signal generated.

In another assay format, one or a combination of at least two monoclonalantibodies can be employed as a competitive probe for the detection ofantibodies to HIV-derived polypeptide protein. For example, HIV-derivedpolypeptide proteins such as the recombinant antigens disclosed herein,either alone or in combination, are coated on a solid phase. A testsample suspected of containing antibody to HIV-derived polypeptideantigen then is incubated with an indicator reagent comprising a signalgenerating compound and at least one monoclonal antibody for a time andunder conditions sufficient to form antigen/antibody complexes of eitherthe test sample and indicator reagent bound to the solid phase or theindicator reagent bound to the solid phase. The reduction in binding ofthe monoclonal antibody to the solid phase can be quantitativelymeasured.

In yet another detection method, each of the monoclonal or polyclonalantibodies can be employed in the detection of HIV-derived polypeptideantigens in fixed tissue sections, as well as fixed cells byimmunohistochemical analysis. Cytochemical analysis wherein theseantibodies are labeled directly (with, for example, fluorescein,colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) orare labeled by using secondary labeled anti-species antibodies (withvarious labels as exemplified herein) may be used to track thehistopathology of disease.

In addition, these monoclonal antibodies can be bound to matricessimilar to CNBr-activated Sepharose and used for the affinitypurification of specific HIV-derived polypeptide proteins from cellcultures or biological tissues such as to purify recombinant and nativeHIV-derived polypeptide antigens and proteins.

Monoclonal antibodies can also be used for the generation of chimericantibodies for therapeutic use, or other similar applications.

The monoclonal antibodies or fragments thereof can be providedindividually to detect HIV-derived polypeptide antigens. Combinations ofthe monoclonal antibodies (and fragments thereof) also may be usedtogether as components in a mixture or “cocktail” of at least oneHIV-derived polypeptide antibody with antibodies to other HIV-derivedpolypeptide regions, each having different binding specificities. Thus,this cocktail can include monoclonal antibodies which are directed toHIV-derived polypeptide proteins of HIV and other monoclonal antibodiesto other antigenic determinants of the HIV-derived polypeptide genome.

The polyclonal antibody or fragment thereof which can be used in theassay formats should specifically bind to an HIV-derived polypeptideregion or other HIV-derived polypeptide proteins used in the assay. Thepolyclonal antibody used preferably is of mammalian origin; human, goat,rabbit or sheep anti-HIV-derived polypeptide polyclonal antibody can beused. Most preferably, the polyclonal antibody is rabbit polyclonalanti-HIV-derived polypeptide antibody. The polyclonal antibodies used inthe assays can be used either alone or as a cocktail of polyclonalantibodies. Since the cocktails used in the assay formats are comprisedof either monoclonal antibodies or polyclonal antibodies havingdifferent HIV-derived polypeptide specificity, they would be useful fordiagnosis, evaluation and prognosis of HIV-derived polypeptidecondition, as well as for studying HIV-derived polypeptide proteindifferentiation and specificity.

It is contemplated and within the scope of the present invention thatHIV-derived polypeptides may be detectable in assays by use ofrecombinant antigens as well as by use of synthetic peptides or purifiedpeptides, which contain amino acid sequences of HIV-derivedpolypeptides. It also is within the scope of the present invention thatdifferent synthetic, recombinant or purified peptides identifyingdifferent epitopes of each such HIV-derived polypeptide can be used incombination in an assay to diagnose, evaluate, or prognosticate the HIVdisease condition. In this case, these peptides can be coated onto onesolid phase, or each separate peptide may be coated on separate solidphases, such as microparticles, and then combined to form a mixture ofpeptides which can be later used in assays. Furthermore, it iscontemplated that multiple peptides which define epitopes from differentpolypeptides may be used in combination to make a diagnosis, evaluation,or prognosis of HIV disease. Peptides coated on solid phases or labeledwith detectable labels are then allowed to compete with peptides from apatient sample for a limited amount of antibody. A reduction in bindingof the synthetic, recombinant, or purified peptides to the antibody (orantibodies) is an indication of the presence of HIV-secretedpolypeptides in the patient sample which in turn indicates the presenceof HIV gene in the patient. Such variations of assay formats are knownto those of ordinary skill in the art and are discussed herein below.

In another assay format, the presence of antigens and/or antibodies toHIV-derived polypeptides can be detected in a simultaneous assay, asfollows. A test sample is simultaneously contacted with a capturereagent of a first analyte, wherein said capture reagent comprises afirst binding member specific for a first analyte attached to a solidphase and a capture reagent for a second analyte, wherein said capturereagent comprises a first binding member for a second analyte attachedto a second solid phase, to thereby form a mixture. This mixture isincubated for a time and under conditions sufficient to form capturereagent/first analyte and capture reagent/second analyte complexes.These so-formed complexes then are contacted with an indicator reagentcomprising a member of a binding pair specific for the first analytelabeled with a signal generating compound and an indicator reagentcomprising a member of a binding pair specific for the second analytelabeled with a signal generating compound to form a second mixture. Thissecond mixture is incubated for a time and under conditions sufficientto form capture reagent/first analyte/indicator reagent complexes andcapture reagent/second analyte/indicator reagent complexes. The presenceof one or more analytes is determined by detecting a signal generated inconnection with the complexes formed on either or both solid phases asan indication of the presence of one or more analytes in the testsample. In this assay format, recombinant antigens may be utilized aswell as monoclonal antibodies produced therefrom. Such assay systems aredescribed in greater detail in EP Publication No. 0473065.

In yet other assay formats, the polypeptides disclosed herein may beutilized to detect the presence of antibodies specific for HIV-derivedpolypeptides in test samples. For example, a test sample is incubatedwith a solid phase to which at least one recombinant protein has beenattached. These are reacted for a time and under conditions sufficientto form antigen/antibody complexes. Following incubation, theantigen/antibody complex is detected. Indicator reagents may be used tofacilitate detection, depending upon the assay system chosen. In anotherassay format, a test sample is contacted with a solid phase to which arecombinant protein produced as described herein is attached and also iscontacted with a monoclonal or polyclonal antibody specific for theprotein, which preferably has been labeled with an indicator reagent.After incubation for a time and under conditions sufficient forantibody/antigen complexes to form, the solid phase is separated fromthe free phase, and the label is detected in either the solid or freephase as an indication of the presence of HIV-derived polypeptideantibody. Other assay formats utilizing the recombinant antigensdisclosed herein are contemplated. These include contacting a testsample with a solid phase to which at least one antigen from a firstsource has been attached, incubating the solid phase and test sample fora time and under conditions sufficient to form antigen/antibodycomplexes, and then contacting the solid phase with a labeled antigen,which antigen is derived from the same source or, alternatively, asecond source different from the first source. For example, arecombinant protein derived from a first source such as E. coli is usedas a capture antigen on a solid phase, a test sample is added to theso-prepared solid phase, and a recombinant protein derived from adifferent source (i.e., non-E. coli) is utilized as a part of anindicator reagent. Likewise, combinations of a recombinant antigen on asolid phase and synthetic peptide in the indicator phase also arepossible. Any assay format which utilizes an antigen specific forHIV-derived polypeptide from a first source as the capture antigen andan antigen specific for HIV-derived polypeptide from a second source arecontemplated. Thus, various combinations of recombinant antigens, aswell as the use of synthetic peptides, purified proteins, and the like,are within the scope of this invention. Assays such as this and othersare described in U.S. Pat. No. 5,254,458, which enjoys common ownershipand is incorporated herein by reference.

Other embodiments which utilize various other solid phases also arecontemplated and are within the scope of this invention. For example,ion capture procedures for immobilizing an immobilizable reactioncomplex with a negatively charged polymer (described in EP publication0326100 and EP publication No. 0406473), can be employed according tothe present invention to effect a fast solution-phase immunochemicalreaction. An immobilizable immune complex is separated from the rest ofthe reaction mixture by ionic interactions between the negativelycharged poly-anion/immune complex and the previously treated, positivelycharged porous matrix and detected by using various signal generatingsystems previously described, including those described inchemiluminescent signal measurements as described in EPO Publication No.0 273,115.

Also, the methods of the present invention can be adapted for use insystems which utilize microparticle technology including in automatedand semi-automated systems wherein the solid phase comprises amicroparticle (magnetic or non-magnetic). Such systems include thosedescribed in published EPO applications Nos. EP 0 425 633 and EP 0 424634, respectively.

The use of scanning probe microscopy (SPM) for immunoassays also is atechnology to which the monoclonal antibodies of the present inventionare easily adaptable. In scanning probe microscopy, in particular inatomic force microscopy, the capture phase, for example, at least one ofthe monoclonal antibodies of the invention, is adhered to a solid phaseand a scanning probe microscope is utilized to detect antigen/antibodycomplexes which may be present on the surface of the solid phase. Theuse of scanning tunneling microscopy eliminates the need for labelswhich normally must be utilized in many immunoassay systems to detectantigen/antibody complexes. The use of SPM to monitor specific bindingreactions can occur in many ways. In one embodiment, one member of aspecific binding partner (analyte specific substance which is themonoclonal antibody of the invention) is attached to a surface suitablefor scanning. The attachment of the analyte specific substance may be byadsorption to a test piece which comprises a solid phase of a plastic ormetal surface, following methods known to those of ordinary skill in theart. Or, covalent attachment of a specific binding partner (analytespecific substance) to a test piece which test piece comprises a solidphase of derivatized plastic, metal, silicon, or glass may be utilized.Covalent attachment methods are known to those skilled in the art andinclude a variety of means to irreversibly link specific bindingpartners to the test piece. If the test piece is silicon or glass, thesurface must be activated prior to attaching the specific bindingpartner. Also, polyelectrolyte interactions may be used to immobilize aspecific binding partner on a surface of a test piece by usingtechniques and chemistries. The preferred method of attachment is bycovalent means. Following attachment of a specific binding member, thesurface may be further treated with materials such as serum, proteins,or other blocking agents to minimize non-specific binding. The surfacealso may be scanned either at the site of manufacture or point of use toverify its suitability for assay purposes. The scanning process is notanticipated to alter the specific binding properties of the test piece.

While the present invention discloses the preference for the use ofsolid phases, it is contemplated that the reagents such as antibodies,proteins and peptides of the present invention can be utilized innon-solid phase assay systems. These assay systems are known to thoseskilled in the art, and are considered to be within the scope of thepresent invention.

The present invention will be better understood in connection with thefollowing examples, which are meant to illustrate, but not to limit, thespirit and scope of the invention.

EXAMPLE 1 Cloning Procedures

Oligonucleotides for gene construction and sequencing were synthesizedat Abbott Laboratories, Synthetic Genetics (San Diego, Calif.) or OligoEtc. (Wilsonville, Calif.). All polymerase chain reaction (PCR)reagents, including AmpliTaq DNA polymerase and UlTma DNA polymerase,were purchased from Perkin-Elmer Corporation (Foster City, Calif.) andused according to the manufacturer's specifications unless otherwiseindicated. PCR amplifications were performed on a GeneAmp 9600 thermalcycler (Perkin-Elmer). Unless indicated otherwise, restriction enzymeswere purchased from New England BioLabs (Beverly, Mass.) and digestswere performed as recommended by the manufacturer. DNA fragments usedfor cloning were isolated on agarose (Life Technologies, Gaithersburg,Md.) gels, unless otherwise indicated.

Desired fragments were excised and the DNA was extracted with a QIAEX IIgel extraction kit or the QIAquick gel extraction kit (Qiagen Inc.,Chatsworth, Calif.) as recommended by the manufacturer. DNA wasresuspended in H₂O or TE [1 mM ethylenediaminetetraacetic acid (EDTA; pH8.0; BRL Life Technologies), 10 mMtris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl; pH 8.0; BRLLife Technologies)]. Ligations were performed using a Stratagene DNAligation kit (Stratagene Cloning Systems, La Jolla, Calif.) asrecommended by the manufacturer. Ligations were incubated at 16° C.overnight.

Bacterial transformations were performed using MAX EFFICIENCY DH5αcompetent cells (BRL Life Technologies) or Epicurian Coli XL1-Bluesupercompetent cells (Stratagene Cloning Systems) following themanufacturer's protocols. Unless indicated otherwise, transformationsand bacterial restreaks were plated on LB agar (Lennox) plates with 150μg/ml ampicillin (M1090; MicroDiagnostics, Lombard, Ill.) or on LBagar+ampicillin plates supplemented with glucose to a finalconcentration of 20 mM, as noted. All bacterial incubations (plates andovernight cultures) were conducted overnight (˜16 hours) at 37° C.

Screening of transformants to identify desired clones was accomplishedby sequencing of miniprep DNA and/or by colony PCR. Miniprep DNA wasprepared with a Qiagen Tip 20 Plasmid Prep Kit or a Qiagen QIAwell 8Plasmid Prep Kit following the manufacturer's specifications, unlessotherwise indicated. For colony PCR screening, individual colonies werepicked from transformation plates and transferred into a well in asterile flat-bottom 96-well plate (Costar, Cambridge, Mass.) containing100 μl sterile H₂O. One-third of the volume was transferred to a secondplate and stored at 4° C. The original 96-well plate was microwaved for5 minutes to disrupt the cells. 1 μl volume then was transferred to aPCR tube as template. 9 μl of a PCR master mix containing 1 μl 10×PCRbuffer, 1 μl 2 mM dNTPs, 1 μl (10 μmol) sense primer, 1 μl (10 μmol)anti-sense primer, 0.08 μl AmpliTaq DNA polymerase (0.4 units), and 4.2μl H₂O was added to the PCR tube. Reactions were generally amplified for20-25 cycles of 94° C. for 30 seconds, 50-60° C. (depending on primerannealing temperatures) for 30 seconds and 72° C. for 60 seconds.Primers were dependent on the insert and cycle conditions were modifiedbased on primer annealing temperatures and the length of the expectedproduct. After cycling, approximately ⅓ of the reaction volume wasloaded on an agarose gel for analysis. Colonies containing desiredclones were propagated from the transfer plate.

Unless otherwise indicated, DNA sequencing was performed on an automatedABI Model 373A Stretch Sequencer (Perkin Elmer). Sequencing reactionswere set up with reagents from a FS TACS Dye Term Ready Reaction Kit(Perkin Elmer) and 250-500 ng plasmid DNA according to themanufacturer's specifications. Reactions were processed on Centri-Sepcolumns (Princeton Separations, Adelphia, N.J.) prior to loading on theSequencer. Sequence data was analyzed using Sequencher 3.0 (Gene CodesCorporation, Ann Arbor, Mich.) and GeneWorks 2.45 (Oxford MolecularGroup, Inc., Campbell, Calif.).

EXAMPLE 2 Determination of the env Sequence of the HIV-1 Group O IsolateHAM112

Viral RNA was extracted from culture supernatants of human peripheralblood mononuclear cells infected with the HIV-1 Group O isolatedesignated HAM112 (H. Hampl et al., Infection 23:369-370 [1995]) using aQIAamp Blood Kit (Qiagen) and the manufacturer's recommended procedure.RNA was eluted in a 50 μl volume of nuclease-free water (5Prime-3Prime,Inc., Boulder, Colo.) and stored at −70° C. The strategy for obtainingthe env region sequence involved cDNA synthesis and PCR (nested)amplification of four overlapping env gene fragments. The amplifiedproducts were sequenced directly on an automated ABI Model 373A StretchSequencer. Amplification reactions were carried out with GeneAmp RNA PCRand GeneAmp PCR Kits (Perkin Elmer) as outlined by the manufacturer.Oligonucleotide primer positions correspond to the HIV-1 ANT70 envsequence (G. Myers et al., eds., supra). The primers env10R [nucleotide(nt) 791-772; SEQ ID NO:62], env15R (nt 1592-1574; SEQ ID NO:63), env22R(nt 2321-2302; SEQ ID NO:64), env26R (nt 250-232 3′ of env; SEQ IDNO:65) were used for cDNA synthesis of fragments 1-4, respectively.Reverse transcription reactions were incubated at 42° C. for 30 minutesthen at 99° C. for 5 minutes. First-round PCR amplifications consistedof 30 cycles of 95° C. for 30 seconds, 52° C. for 30 seconds, and 72° C.for 1 minute using the primer combinations: env1F (nt 184-166 5′ of env;SEQ ID NO:66) and env10R (SEQ ID NO:62), env7F (nt 564-586; SEQ IDNO:67) and env15R (SEQ ID NO:63), env12F (nt 1289-1308; SEQ ID NO:68)and env22R (SEQ ID NO:64), env19F (nt 2020-2040; SEQ ID NO:69) andenv26R (SEQ ID NO:65) for fragments 1 through 4, respectively. For thesecond round of amplification (nested PCR), 5 μl of the respectivefirst-round PCR reactions was used as template along with the primercombinations env2F (nt 37-15 5′ of env; SEQ ID NO:70) and env9R (nt740-721; SEQ ID NO:71), env8F (nt 631-650; SEQ ID NO:72) and env14R (nt1437-1416; SEQ ID NO:73), env13F (nt 1333-1354; SEQ ID NO:74) and env21R(nt 2282-2265; SEQ ID NO:75), env20F (nt 2122-2141; SEQ ID NO:76) andenv25R (nt 111-94 3′ of env; SEQ ID NO:77) for fragments 1 through 4,respectively. Second-round amplification conditions were identical tothose used for the first round. Fragments were agarose gel-purified andextracted with a Qiagen QIAEX II Gel Extraction Kit. Fragments weresequenced directly with the primers used for nested PCR along withprimers env4F (SEQ ID NO:78) and env5R (SEQ ID NO:79) for fragment 1;primers env10F (SEQ ID NO:80), env11F (SEQ ID NO:81), env11R (SEQ IDNO:82), env12F (SEQ ID NO:68), and AG1 (SEQ ID NO:87) for fragment 2;primers env15F (SEQ ID NO:83) and env19R (SEQ ID NO:84) for fragment 3;primers env22F (SEQ ID NO:85) and env24R (SEQ ID NO:86) for fragment 4.The deduced amino acid sequence of env from the HIV-1 Group O isolateHAM112 is shown in SEQ ID NO:61.

EXAMPLE 3 Construction of Synthetic HIV-1 Group O env gp120/gp41 Genes

Synthetic HIV-1 Group O env gp120/gp41 gene constructs were generated.The env gp120/gp41 sequences were based on the HIV-1 Group O isolateHAM112 (SEQ ID NO:61). Determination of the env sequence of HAM112 isoutlined in Example 2, hereinabove. Oligonucleotides were designed thatencode the C-terminal 45 amino acids of the env gp120 and 327 aminoacids of env gp41 (nucleotide #1 is the first base of the first codon ofgp120 in the synthetic gene). The synthetic gene has a 26 amino aciddeletion (nucleotides 643 through 720), relative to the native HAM112gp41, that encompasses a highly hydrophobic (H) region (transmembraneregion) of gp41. Thus, the full-length synthetic gp41 gene constructedis 327 amino acids.

In the synthetic oligonucleotides, the native HIV-1 codons were alteredto conform to E. coli codon bias in an effort to increase expressionlevels of the recombinant protein in E. coli. See, for example, M. Gouyand C. Gautier, Nucleic Acids Research 10:7055 (1982); H. Grosjean andW. Fiers, Gene 18:199 (1982); J. Watson et al. (eds.), Molecular Biologyof the Gene, 4th Ed., Benjamin Kumming Publishing Co., pp. 440 (1987).The gene construction strategy involved synthesis of a series ofoverlapping oligonucleotides with complementary ends (Osyn-A throughOsyn-L, depicted as A through L). When annealed, the ends served asprimers for the extension of the complementary strand.

The fragments then were amplified by PCR. This process (“PCR knitting”of oligonucleotides) was reiterated to progressively enlarge the genefragment. Oligonucleotide Osyn-5′ was designed for cloning into the PLvector pKRR826. The expression vector, pKRR826, is a modified form ofthe lambda pL promoter vector pSDKR816, described in U.S. Ser. No.08/314,570, incorporated herein by reference. pKRR826 is a high copynumber derivative of pBR322 that contains the temperature sensitive cIrepressor gene (Benard et al., Gene 5:59 [1979]). However, pKRR826 lacksthe translational terminator rmBt1 and has the lambda pL and lambda pRpromoters in the reverse orientation, relative to pSDKR816. Thepolylinker region of pKRR826 contains Eco RI and Bam HI restrictionenzyme sites but lacks an ATG start codon. Optimal expression isobtained when the 5′ end of the gene insert (including an N-terminalmethionine) is cloned into the EcoRI site. Osyn-5′ was designed tocontain an Eco RI restriction site for cloning and an ATG codon(methionine) to provide for proper translational initiation of therecombinant proteins. The anti-sense oligonucleotides Osyn-O3′ (SEQ IDNO:15), Osyn-P3′ (SEQ ID NO:16), and Osyn-M (M) (SEQ ID NO:14) eachcontain two sequential translational termination codons (TAA,TAG) and aBam HI restriction site. When outside primers Osyn-5′ (SEQ ID NO:11) andOsyn-M (M) (SEQ ID NO:14) were used, a full-length gp41 (327 aminoacids) gene was synthesized (pGO-11PL; SEQ ID NO:52). Outsideoligonucleotides Osyn-5′ (SEQ ID NO:11) and Osyn-P3′ (SEQ ID NO:15)resulted in a truncated gp41 product of 199 amino acids (pGO-9PL; SEQ IDNO:48). Alternatively, outside oligonucleotides Osyn-5′ (SEQ ID NO:11)and Osyn-P3′ (SEQ ID NO:16) resulted in a truncated gp41 product 169amino acids in length (pGO-8PL; SEQ ID NO:58).

The synthetic genes also were expressed as CMP-KDO synthetase (CKS)fusion proteins. PCR-mediated transfer of the synthetic genes frompKRR826 into pJO200 (described in U.S. Serial No. 572,822, andincorporated herein by reference) was accomplished with an alternativeoutside sense oligonucleotide PCR primer (5′ end), Osyn-5′CKS (SEQ IDNO:25). Osyn-5′CKS contained an Eco RI restriction site and resulted inthe in-frame fusion of the synthetic gene insert to CKS in theexpression vector pJO200. The 3′ outside primers (antisense) Osyn-M (SEQID NO:14), Osyn-O3′ (SEQ ID NO:15) and Osyn-P3′ (SEQ ID NO:16) were usedin combination with Osyn-5′CKS (SEQ ID NO:25) to generate pGO-11CKS (SEQID NO:54), pGO-9CKS (SEQ ID NO:50), and pGO-8 CKS (SEQ ID NO:60),respectively. These steps are detailed hereinbelow.

A. PCR Knitting of Synthetic Oligonucleotides.

Three PCR reactions (100 μl volume) were set up as follows:

(1) Reaction 1B: AmpliTaq DNA polymerase (2.5 U) and 1× buffer, alongwith 40 μM of each dNTP (dATP, dCTP, dGTP, and dTTP), 25 μmol each ofoligonucleotides Osyn-A (SEQ ID NO:3) and Osyn-D (SEQ ID NO:5), and 0.25μmol each of oligonucleotides Osyn-B (SEQ ID NO:17) and Osyn-C (SEQ IDNO:4);

(2) Reaction 2A: UlTma DNA Polymerase (3 U) and 1× buffer along with 1.5mM MgCl₂, 40 μM of each dNTP, 25 μmol each of oligonucleotides Osyn-E(SEQ ID NO:6) and Osyn-H (SEQ ID NO:9), and 0.25 μmol each ofoligonucleotides Osyn-F (SEQ ID NO:7) and Osyn-G (SEQ ID NO:8); and

(3) Reaction 3A: UlTma DNA Polymerase (3 U) and 1× buffer along with 1.5mM MgCl₂, 40 μM of each dNTP, 25 μmol each of oligonucleotides Osyn-I(SEQ ID NO:10) and Osyn-L (SEQ ID NO:13), and 0.25 μmol each ofoligonucleotides Osyn-J (SEQ ID NO:18) and Osyn-K (SEQ ID NO:12).

Amplifications consisted of 20 cycles of 97° C. for 30 seconds, 52° C.for 30 seconds and 72° C. for 60 seconds. Reactions were then incubatedat 72° C. for 7 minutes and held at 4° C. PCR-derived products 1B, 2Aand 3B were gel isolated on a 1% agarose gel.

B. PCR Knitting of PCR Products from Reaction 1B and Reaction 2A.

A PCR reaction was set up with UlTma DNA Polymerase (3 U) and 1× bufferalong with 1.5 mM MgCl₂, 40 μM of each dNTP, 24.4 μmol ofoligonucleotide Osyn-5′ (SEQ ID NO:11), 25 μmol of oligonucleotideOsyn-P3′ (SEQ ID NO:16), and ˜10 ng each of gel-isolated 1B and 2Aproducts from Example 3, Section 1A, hereinabove. Cycling conditionswere the same as in Example 3, Section 1A. A second round ofamplification was used to generate more of the desired product. This wasperformed by making an UlTma mix as described hereinabove (100 μlreaction volume) with 49 μmol Osyn-5′ (SEQ ID NO:11), 50 μmol Osyn-P3′(SEQ ID NO:16) and 5 μl of the PCR product from the first round astemplate. These reactions were incubated at 94° C. for 90 seconds, andthen cycled as above (Section 3A). The Osyn-5′/Osyn-P3′ PCR product wasgel-isolated as described hereinabove.

C. Cloning of the Osyn-5′-Osyn-P3′ PCR Product.

The Osyn-5′-Osyn-P3′ PCR product was digested with the restrictionendonucleases Eco RI+Bam HI and ligated into the vector pKRR826(described hereinabove) that had been digested with Eco RI+Bam HI andgel-isolated. The ligation product was used to transform DH5α competentcells. The desired clone was identified by colony PCR usingoligonucleotides pKRREcoRI Forward (SEQ ID NO:38) and pKRRBamHI Reverse(SEQ ID NO:39). Miniprep DNA was prepared from an overnight culture ofpGO-8 candidate clone A2 and the Osyn-5′-Osyn-P3′ plasmid insert wassequenced with the oligonucleotide primers pKRREcoRI Forward (SEQ IDNO:38), pKRRBamHI Reverse (SEQ ID NO:39), 41sy-1 (SEQ ID NO:44), and41sy-2 (SEQ ID NO:41).

D. Modification of pGO-8 Candidate Clone A2.

A 100 μl volume PCR reaction was set up with UlTma DNA Polymerase (3 U)and 1× buffer, along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 μmol ofoligonucleotides Osyn-5′-repair (SEQ ID NO:24), 50 μmol Osyn-P3′ (SEQ IDNO:16), and ˜1 ng of pGO-8 candidate clone A2 miniprep DNA as template(obtained from the reactions set forth hereinabove). The reaction wasincubated at 94° C. for 90 seconds, and then amplified with 20 cycles of94° C. for 30 seconds, 50° C. for 30 seconds and 72° C. for 60 seconds.The Osyn-5′-repair/Osyn-P3′ PCR product then was gel isolated anddigested with Eco RI+Bam HI. The digested product was ligated into EcoRI+Bam HI digested pKRR826 vector. The ligation product was used totransform DH5α competent cells. The desired clone was identified bycolony PCR using oligonucleotides pKRREcoRI Forward (SEQ ID NO:38) andpKRRBamHI Reverse (SEQ ID NO:39). An overnight culture of pGO-8candidate clone 6 was set up and a miniprep DNA was prepared. TheOsyn-5′repair/Osyn-P3′ plasmid insert was sequenced with theoligonucleotide primers pKRREcoRI Forward (SEQ ID NO:38), pKRRBamHIReverse (SEQ ID NO:39), 41sy-1 (SEQ ID NO:44), and 41sy-2 (SEQ IDNO:41). Based on the sequencing results, pGO-8 candidate clone #6 wasdesignated pGO-8PL/DHSα. SEQ ID NO:57 presents the nucleotide sequenceof the coding region. SEQ ID NO:58 presents the amino acid sequence ofthe pGO-8PL recombinant protein. The pGO-8PL recombinant proteinconsists of a N-terminal methionine, 45 amino acids gion of env gp120(HIV-1 Group O, HAM112 isolate), and 169 amino acids of env gp41 (HIV-1Group O, HAM112 isolate).

E. Construction of pGO-8CKS/XL1.

pGO-8CKS/XL1 (SEQ ID NO:59) presents the nucleotide sequence of thecoding region) encodes the recombinant protein pGO-8CKS. SEQ ID NO:60 isthe amino acid sequence of pGO-8CKS. This protein consists of 246 aminoacids of CKS/polylinker, 45 amino acids of env gp120 (HIV-1 Group O,HAM112 isolate), and 169 amino acids of env gp41 (HIV-1 Group O, HAM112isolate). The construction of pGO-8CKS/XL1 was accomplished as follows.

A PCR reaction (100 μl volume) was set up with UlTma DNA Polymerase (3U) and 1× buffer along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 μmol ofOsyn-5′CKS (SEQ ID NO:25), 50 μmol Osyn-P3′ (SEQ ID NO: 16), and 1 ngpGO-8PL clone #6 miniprep DNA. The reaction was incubated at 94° C. for90 seconds then amplified with 25 cycles of 94° C. for 30 seconds; 55°C. for 30 seconds; 72° C. for 90 seconds. Then, the Osyn-5′CKS/Osyn-P3′PCR product was gel isolated. EcoR I+Bam HI digested theOsyn-5′CKS/Osyn-P3′ PCR product and the vector pJO200. The digestedpJO200 vector was gel isolated and ligated to digestedOsyn-5′CKS/Osyn-P3′ PCR product. XL1-Blue supercompetent cells weretransformed with the ligation and plated on LB+ampicillin platessupplemented with 20 mM glucose. Colonies were restreaked for isolationon the same type of plates. An overnight culture of clone pGO-8CKS/XL1was grown in LB broth+100 μg/ml carbenicillin (Sigma Chemical Co.)+20 mMglucose (Sigma Chemical Co.). Frozen stocks (0.5 ml overnightculture+0.5 ml glycerol) were made and DNA was prepared for sequenceanalysis. The following oligonucleotides were used as sequencingprimers: CKS-1 (SEQ ID NO:30), CKS-2 (SEQ ID NO:31), CKS-3 (SEQ IDNO:32), CKS-4 (SEQ ID NO:33), 43461 (SEQ ID NO:2), 43285 (SEQ ID NO:1),41sy-1B (SEQ ID NO:29), 41sy-2B (SEQ ID NO:34), CKS176.1 (SEQ ID NO:19),and CKS3583 (SEQ ID NO:20).

F. Construction of pGO-9PL/DH5α.

The construct pGO-9PL/DH5α encodes the recombinant protein pGO-9PL. SEQID NO:47 is the nucleotide sequence of the coding region of pGO-9PL/DH5αand SEQ ID NO: 48 is the amino acid sequence of the pGO-9PL recombinantprotein. This protein consists of an N-terminal methionine, 45 aminoacids of env gp120 (HIV-1 Group O, HAM112 isolate), and 199 amino acidsof env gp41 (HIV-1 Group O, HAM112 isolate). Construction ofpGO-9PL/DH5α was accomplished as follows.

Step 1: A 100 μl PCR reaction was set up with UlTma DNA Polymerase (3 U)and 1× buffer, along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 μmol ofOsyn-5′ (SEQ ID NO:11), 50 μmol of Osyn-H (SEQ ID NO:9), and ˜2 ng ofpGO-8 candidate clone 6 miniprep DNA (obtained from Example 3, Section Dhereinabove) as template. The reaction was incubated at 94° C. for 120seconds, and then amplified with 8 cycles of 94° C. for 30 seconds, 55°C. for 30 seconds and 72° C. for 60 seconds.

Step 2: A 100 μl PCR reaction was set up with UlTma DNA Polymerase (3 U)and 1× buffer along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 μmol ofOsyn-5′ (SEQ ID NO:11), 50 μmol Osyn-O3′ (SEQ ID NO:15), and 10 μl ofthe PCR reaction from step 1 as template. The reaction was incubated at94° C. for 120 seconds then amplified with 18 cycles of 94° C. for 30seconds, 55° C. for 30 seconds, 72° C. for 60 seconds, followed byincubation at 72° C. for 5 minutes.

The Osyn-5′/Osyn-O3′ PCR product (2A/2B) then was gel-isolated anddigested with Eco RI+Bam HI. The digested product was ligated into EcoRI+Bam HI digested pKRR826 vector. The ligation product next was used totransform DH5α competent cells. An overnight culture of pGO-9PLcandidate clone 3 was set up and a miniprep DNA was prepared. TheOsyn-5′/Osyn-O3′ plasmid insert was sequenced with the followingoligonucleotides as primers: pKRREcoR1 Forward (SEQ ID NO:38), pKRRBamHIReverse (SEQ ID NO:39), 41sy-1C (SEQ ID NO:40), 41sy-2 (SEQ ID NO:41),41sy-3 (SEQ ID NO:42) and 41sy-4 (SEQ ID NO:23). pGO-9PL clone #3 thenwas restreaked for isolation. An isolated colony was picked, anovernight culture of it was grown, and a frozen stock (0.5 mlglycerol+0.5 ml overnight culture) was made. The stock was stored at−80° C. The sequence was confirmed using the primers indicatedhereinabove, and this clone was designated as pGO-9PL/DH5α (SEQ ID NO:47presents the nucleotide sequence of the coding region, and SEQ ID NO:48presents the amino acid sequence of coding region). pGO-9PL/DH5α wasrestreaked, an overnight culture was grown, and a miniprep DNA wasprepared (this prep was designated as H5).

G. Construction of pGO-9CKS/XL1

The construct pGO-9CKS/XL1 encodes the recombinant protein pGO-9CKS. SEQID NO:50 is the amino sequence of the pGO-9CKS recombinant protein. Thisprotein consists of 246 amino acids of CKS and polylinker followed by 45amino acids of env gp120 (HIV-1 Group O, HAM112 isolate), and 199 aminoacids of env gp41 (HIV-1 Group O, HAM112 isolate). The construction ofpGO-9CKS/XL1 was accomplished as follows.

Two PCR reactions (100 μl volume) were set up with UlTma DNA Polymerase(3 U) and 1× buffer, along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50μmol of Osyn-5′CKS (SEQ ID NO:25), 50 μmol Osyn-O3′ (SEQ ID NO:15) and 1ng pGO-9PL candidate clone 3 miniprep DNA (obtained from Example 3,Section F, hereinabove). Each reaction was incubated at 94° C. for 120seconds, then amplified with 24 cycles of 94° C. for 30 seconds, 55° C.for 30 seconds, 72° C. for 120 seconds, followed by incubation at 72° C.for 5 minutes. The Osyn-5′CKS/Osyn-O3′ PCR product then was gelisolated. The Osyn-5′CKS/Osyn-O3′ PCR product and the vector pJO200 wasdigested with EcoR I+Bam HI. The digested pJO200 vector was gel isolatedand ligated to the digested Osyn-5′CKS/Osyn-O3′ PCR product. XL1-Bluesupercompetent cells were transformed with the ligation and plated onLB+ampicillin plates supplemented with 20 mM glucose. Colonies wererestreaked for isolation on the same type of plates. An overnightculture of clone pGO-9CKS candidate clone 4 was grown in LB broth+100mg/ml carbenicillin (Sigma Chemical Co.)+20 mM glucose (Sigma ChemicalCo.). Made frozen stocks (0.5 ml overnight culture+0.5 ml glycerol) andprepared DNA for sequence analysis. The following oligonucleotides wereused as sequencing primers: CKS-1 (SEQ ID NO:30), CKS-2 (SEQ ID NO:31),CKS-3 (SEQ ID NO:32), CKS-4 (SEQ ID NO:33), 43461 (SEQ ID NO:2), 43285(SEQ ID NO:1), 41sy-1B (SEQ ID NO:29), 41sy-2B (SEQ ID NO:34), 41sy-3B(SEQ ID NO:35), CKS176.1 (SEQ ID NO:19), CKS3583 (SEQ ID NO:20), andpTB-S8 (SEQ ID NO:28). Clone pGO-9CKS candidate clone 4 was designatedas pGO-9CKS/XL1 (SEQ ID NO:49 presents the nucleotide sequence of codingregion, and SEQ ID NO:50 presents the amino acid sequence of codingregion).

H. Construction of Osyn I-M Fragment.

The Osyn-O-M fragment was constructed as follows. A 100 μl PCR reactionwas set up using AmpliTaq DNA Polymerase (2.5 U), 1× buffer, 50 μM ofeach dNTP, 50 μmol I-PCR (SEQ ID NO:26), 50 μmol Osyn-M (SEQ ID NO:14)and 10 ng of gel-isolated PCR fragment 3A (Example 3, section A,hereinabove). The reaction was incubated at 95° C. for 105 seconds, andthen it was amplified with 15 cycles of 95° C. for 30 seconds, 55° C.for 30 seconds, 72° C. for 60 seconds, and then it was held at 72° C.for 7 minutes. The product, designated as Osyn I-M, was gel-isolated andcloned into the PCR II vector (TA Cloning Kit; Invitrogen, San Diego,Calif.) following the manufacturer's recommended procedure. Theresulting ligation product was used to transform DH5α competent cells.Plasmid miniprep DNA was generated from an overnight culture of cloneIM-6, and the gene insert was sequenced with oligonucleotides 56759(SEQUENCE ID NO: 45) and 55848 (SEQ ID NO:46).

I. Synthesis and Knitting of PCR Fragments I/6R and IM-6F.

These procedures were performed as follows.

Step 1: The following PCR reactions (100 μl volume) were set up: (a)I/6R with AmpliTaq DNA Polymerase (2.5 U), 1× buffer, 50 μM of eachdNTP, 50 μmol I-PCR (SEQ ID NO:26), 50 μmol IM-6R (SEQ ID NO:22) and 281ng of clone IM-6 (obtained from Example 3, Section H) as template; (b)6F/M with AmpliTaq DNA Polymerase (2.5 U), 1× buffer, 50 μM of eachdNTP, 50 μmol IM-6F (SEQ ID NO:21), 50 μmol M-PCR (SEQ ID NO:27) and 281ng of clone IM-6 (obtained from Example 3, Section H) as template.

The reactions were incubated at 95° C. for 105 seconds, and thenamplified with 20 cycles of 94° C. for 15 seconds, 60° C. for 30seconds, 72° C. for 60 seconds, then incubated at 72° C. for 7 minutes.The PCR products I/6R and 6F/M next were gel isolated following theprocedures as described hereinabove.

Step 2: A PCR reaction (100 μl volume) was set up with UlTma DNAPolymerase (3 U) and 1× buffer along with 1.5 mM MgCl₂, 40 μM of eachdNTP, 50 μmol of I-PCR (SEQ ID NO:26), 50 μmol M-PCR (SEQ ID NO:27), ˜50ng I/6R, and ˜20 ng 6F/M. The reaction was incubated at 95° C. for 105seconds, and then it was amplified with 20 cycles of 94° C. for 15seconds, 55° C. for 30 seconds, 72° C. for 60 seconds, followed byincubation at 72° C. for 7 minutes. The PCR product was processed on aCentri-sep column (Princeton Separations) following the manufacturer'sinstructions.

J. Construction of pGO-11PL/DH5α.

The construct pGO-11PL/DH5α encodes the recombinant protein pGO-11PL.SEQ ID NO:52 is the amino acid sequence of the pGO-11PL recombinantprotein. This protein consists of an N-terminal methionine, 45 aminoacids of env gp120 (HIV-1 Group O, HAM112 isolate), and 327 amino acidsof env gp41 (HIV-1 Group O, HAM112 isolate). pGO-11PL/DH5α wasconstructed as follows.

The final PCR product from Example 3, Section I and pGO-9PL vector(miniprep H5 from Example 3, section F) were digested sequentially withAge I and Bam HI. The digested pGO-9PL was then treated with calfintestinal alkaline phosphatase (BRL Life Technologies) for 15 minutesat 37° C., phenol/chloroform extracted, and precipitated with NaOAc andEtOH. The vector (pGO-9PL) was subsequently gel-isolated. The digestedpGO-9PL and the digested PCR product were ligated, and the ligationproduct was used to transform DH5α competent cells. Colonies wererestreaked for isolation. Clone pGO11-4 then was identified andrestreaked for isolation. An overnight culture of pGO11-4 was preparedin order to generate frozen stocks and perform miniprep DNA forsequencing. Clone pGO11-4 was sequenced with the followingoligonucleotide primers: pKRREcoR1 Forward (SEQ ID NO:38), pKRRBamHIReverse (SEQ ID NO:39), 41sy-1C (SEQ ID NO:40), 41sy-2 (SEQ ID NO:41),41sy-3 (SEQUENCE ID NO: 42), 41sy-4 (SEQ ID NO:23), 41sy-5B (SEQ IDNO:43), 41sy-5C (SEQ ID NO:36) and 41sy-6B (SEQ ID NO:37). Based on thesequencing results, this clone was designated as pGO-11PL/DH5α (SEQ IDNO:51 presents the nucleotide sequence of the coding region, and SEQ IDNO:52 presents the amino acid sequence of coding region).

K. Construction of pGO-11CKS/XL1.

The construct pGO-11CKS/XL1 encodes the recombinant protein pGO-11CKS.SEQ ID NO:54 is the amino sequence of the pGO-11CKS recombinant protein.This protein consists of 246 amino acids of CKS and polylinker followedby 45 amino acids of env gp120 (HIV-1 Group O, HAM112 isolate), and 327amino acids of env gp41 (HIV-1 Group O, HAM112 isolate). pGO-11CKS/XL1was constructed as follows.

A PCR reaction (100 μl volume) was set up with UlTma DNA Polymerase (3U) and 1× buffer along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 μmol ofOsyn-5′CKS (SEQ ID NO:25), 50 μmol Osyn-M (SEQ ID NO:14), and 1 ngpG011-4 (obtained from Example 3, Section J) as template. The reactionwas incubated at 94° C. for 105 seconds, and then amplified with 20cycles of 94° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 120seconds, followed by incubation at 72° C. for 7 minutes. TheOsyn-5′CKS/Osyn-M PCR product was gel isolated. Next, theOsyn-5′CKS/Osyn-M PCR product and the vector pJO200 were EcoR I+Bam HIdigested. The digested pJO200 vector was gel isolated. Overnight (16°C.) ligations were set up with the digested PCR product. XL1-Bluesupercompetent cells were transformed with the ligation and plated onLB+ampicillin plates supplemented with 20 mM glucose. Colonies wererestreaked for isolation on the same plates. An overnight culture (LBmedium+100 μg/ml carbenicillin+20 mM glucose) of clone pGO-11CKS clonecandidate 2 then was set up. Frozen stocks (0.5 ml 80% glycerol+0.5 mlovernight culture) were made as well as miniprep DNA for sequencing. Thefollowing oligonucleotides were used as primers for sequence analysis:CKS-1 (SEQ ID NO:30), CKS-2 (SEQ ID NO:31), CKS-3 (SEQ ID NO:32), CKS-4(SEQ ID NO:33), 43461 (SEQ ID NO:2), 43285 (SEQ ID NO:1), 41sy-1B (SEQID NO:29), 41sy-2B (SEQ ID NO:34), 41sy-3B (SEQ ID NO:35), 41sy-4 (SEQID NO:23), 41sy-5C (SEQ ID NO:36), 41sy-6B (SEQ ID NO:37), CKS176.1 (SEQID NO:19), CKS3583 (SEQ ID NO:20), and pTB-S8 (SEQ ID NO:28). pGO-11 CKSclone #2 was designated as pGO-11CKS/XL1. SEQ ID NO:53 presents thenucleotide sequence of the coding region of pGO-11CKS/XL1, and SEQ IDNO:54 presents the amino acid sequence of the coding region ofpGO-11CKS/XL1.

EXAMPLE 4 Construction of pHIV210/XL1-Blue

SEQ ID NO:55 is the amino acid sequence of the pHIV-210 recombinantprotein. This protein consists of 247 amino acids of CKS/linkersequences, 60 amino acids from env gp120 (#432-491; HIV-2 isolateD194.10), and 159 amino acids of env gp36 (#492-650; HIV-2 isolateD194.10). The construction of pHIV210/XL1-Blue was accomplished asfollows.

The genomic DNA of HIV-2 isolate D194.10 [H. Kuhnel et al., NucleicAcids Research 18: 6142 (1990)] was cloned into the EMBL3 lambda cloningvector. See H. Kuhnel et al., Proc. Nat'l. Acad. Sci. USA 86: 2383-2387(1989), and H. Kuhnel et al., Nucleic Acids Research 18: 6142 (1990),incorporated herein by reference. The lambda clone containing D194.10(lambda A10) was obtained from Diagen Corporation (Düsseldorf, Germany).A PCR reaction (100 μl volume) was set up using AmpliTaq DNA polymerase(3.75 units), 200 μM each dATP, dCTP, dGTP, and dTTP, 0.5 μg primer 3634(SEQ ID NO:88; annealing to positions 7437-7455 on the HIV-2 isolateD194.10 (EMBL accession #X52223), 0.5 μg primer 3636 (SEQ ID NO:89,annealing to positions 8095-8077), 1×PCR buffer, and 5 μl of the lambdaA10 DNA diluted 1:50. The reaction was incubated 5 minutes at 94° C.then amplified with 35 cycles of 94° C. for 1 minute, 45° C. for 1minute, 72° C. for 2 minutes; followed by an incubation at 72° C. for 5minutes. The PCR reaction was extracted with phenol/chloroform(Boehringer Mannheim Corporation, Indianapolis, Ind.) and the DNA wasethanol (AAPER Alcohol & Chemical Company, Shelbyville, Ky.)precipitated. The DNA was digested with EcoRI+Bam HI and gel purified onan 1.5% agarose gel (SeaKem GTG agarose, FMC Corporation, Rockland,Me.). The purified product was ligated into EcoRI+Bam HI digested pJO200vector using 800 units of T4 DNA ligase (New England BioLabs). XL1-Bluesupercompetent cells (Stratagene) were transformed with 2 μl of theligation as outlined by the manufacturer and plated on LB platessupplemented with ampicillin (Sigma Chemical Company). Overnightcultures were established by inoculating single colonies into SuperbrothII media (GIBCO BRL, Grand Island, N.Y.) supplemented with 50 μg/mlampicillin (Sigma) and 20 mM glucose (Sigma). Frozen stocks wereestablished by adding 0.3 ml of 80% glycerol to 0.7 ml of overnight.After mixing stocks were stored at −70° C. Miniprep DNA was preparedfrom the overnight cultures using the alkaline lysis method followed byPEG precipitation. Sequence reactions were performed with a 7-deaza-dGTPReagent Kit with Sequenase Version 2.0 (United States BiochemicalCorporation, Cleveland, Ohio) as outlined by the manufacturer. Reactionswere run on 6% acrylamide gels (GIBCO BRL Gel-Mix 6) using the IBI gelapparatus as recommended by the manufacturer. Based on sequencingresults, pHIV-210 clone #7 was designated as pHIV-210. The amino acidsequence of the pHIV-210 coding region is presented as SEQ ID NO:55.

EXAMPLE 5 Growth and Induction of E. coli Strains with HIV-1 Group ORecombinant gp41 Antigen Construct

Overnight seed cultures of pGO-9CKS/XL1 and pGO-11CKS/XL1 were preparedin 500 ml sterile Excell Terrific Broth (available from Sigma ChemicalCorp., St. Louis Mo.) supplemented with 100 μg/ml sodium ampicillin, andplaced in a shaking orbital incubator at 32° C. or 37° C. One hundredmilliliter (100 μl) inocula from seed cultures were transferred toflasks containing 1 liter sterile Excell Terrific Broth supplementedwith 100 μg/ml sodium ampicillin. Cultures were incubated at 37° C.until the culture(s) reached mid-logarithmic growth and then inducedwith 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37° C. (In thecase of PL vector constructs, cultures were incubated at 32° C. untilthe culture(s) reached mid-logarithmic growth and then induced for 3hours by shifting the temperature of the culture(s) to 42° C.) After theinduction period, cells were pelleted by centrifugation and harvestedfollowing standard procedures. Pelleted cells were stored at −70° C.until further processed.

EXAMPLE 6 Isolation and Solubilization of HIV-1 Group O Recombinant gp41Antigen Produced as Insoluble Inclusion Bodies in E. coli

Frozen cells obtained from Example 5 were resuspended by homogenizationin cold lysis buffer comprising 50 mM Tris pH 8, 10 mM Na EDTA, 150 mMNaCl, 8% (w/v) sucrose, 5% Triton X-1000 (v/v), 1 mM PMSF and 1 mMpepstatin A. Lysozyme was added to the homogenates at a concentration of1.3 mg per gram of cells processed, and the resultant mixture wasincubated for 30 minutes on ice to lyse the cells. Inclusion bodies wereseparated from soluble proteins by centrifugation. These pelletedinclusion bodies were washed and pelleted sequentially in (1) LysisBuffer; (2) 10 mM Na EDTA pH 8, 30% (w/v) sucrose; and (3) water. Thewashed inclusion bodies were resuspended in 50 mM Tris pH 8, 10 mM NaEDTA, 150 mM NaCl and 3 M urea, and incubated on ice for 1 hour. Theinclusion bodies then were separated from the solubilized proteins bycentrifugation. The pelleted inclusion bodies were fully solubilized in7 M guanidine-HCl, 50 mM Tris pH 8, 0.1% (v/v) beta-mercaptoethanol(BME) overnight at 4° C. The solubilized recombinant antigens wereclarified by centrifugation, passed through a 0.2 μm filter and storedat ≦−20° C. until purified by chromatography.

EXAMPLE 7 Purification of Recombinant HIV-1 Group O gp41 Antigen byChromatography

Solubilized HIV-1 Group O recombinant gp41 antigens obtained fromExample 6 were purified by a two-step method, as follows. Guanidine-HClextracts of insoluble antigens were purified by size exclusionchromatography on a Sephacryl S-300 column equilibrated with 50 mM TrispH 8, 8 M Urea and 0.1% BME (v/v). SDS-polyacrylamide electrophoresiswas used to analyze fractions. Fractions containing the recombinant gp41antigen were pooled and then concentrated by ultrafiltration. Therecombinant antigen concentrate was treated with 4% SDS (w/v) and 5% BME(w/v) at room temperature for 3 hours. SDS treated antigen was furtherpurified by size exclusion chromatography on a Sephacryl S-300 columnequilibrated with 25 mM Tris pH 8, 0.15 M NaCl, 0.1% v/v BME, 0.1% SDS(w/v). SDS-polyacrylamide electrophoresis was used to analyze thefractions. Fractions containing purified recombinant antigen werepooled, passed through a 0.2 μm filter and stored at −70° C.

EXAMPLE 8 Preparation of HIV-1 Group M Antigen

Cells containing the plasmid pTB319 were grown and induced as describedin Example 5. Cells were lysed and inclusion bodies were processedessentially as described in Example 5 of U.S. Pat. No. 5,124,255,incorporated herein by reference. The pellet material was subsequentlysolubilized in SDS, Phosphate, pH 6.8 and then subjected tochromatography on an S-300 column.

EXAMPLE 9 Preparation of HIV-2 Antigen

pHIV-210/XL1-Blue cells (Example 4, hereinabove) were grown and inducedas described in Example 5. Cells were lysed with a buffer containingphosphate, MgCl₂, Na EDTA, Triton X-100® pH 7.4 supplemented withBenzonase, Lysozyme, and PMSF. Inclusion bodies were separated fromsoluble proteins by centrifugation. The pellet was washed sequentiallywith: distilled H₂O; Triton X-100®, deoxycholate, NaCl, Phosphate pH7.0; 50 mM Phosphate, pH 7.0; urea, SDS in phosphate, pH 7.0+BME.Proteins were solubilized in SDS, phosphate, pH 7.0 and BME thensubjected to chromatography on an S300 column.

EXAMPLE 10 One-Step Immunochromatographic Assay for SimultaneousDetection and Differentiation of HIV-1 Group M, HIV-1 Group O and HIV-2

A. Reagent Preparation

1. A selenium (Se) colloid suspension was prepared substantially asfollows: SeO₂ was dissolved in water to a concentration of 0.0625 gm/ml.Ascorbate then was dissolved in water to a concentration of 0.32 gm/mland heated in a 70° C. water bath for 24 hours. The ascorbate solutionthen was diluted to 0.0065 gm/ml in water. The SeO₂ solution was quicklyadded to the diluted ascorbate solution and incubated at 42° C.Incubation was ended after a minimum of 42 hours when the absorbancemaximum exceeded 30 at a wavelength between 542 nm and 588 nm. Thecolloid suspension was cooled to 2-8° C., then stored. Selenium colloidsuspension is available from Abbott Laboratories, Abbott Park, Ill.(Code 25001).

2. Selenium colloid/antibody conjugates were prepared as follows. Theselenium colloid suspension was concentrated to an absorbance of 25 (OD500-570) in distilled water. Then, 1M MOPS was added to a finalconcentration of 10 mM pH 7.2. Goat antibodies specific for human IgG Fcregion (or other species of antibody specific for human IgG Fc region)were diluted to a concentration of 0.75 mg/ml with 50 mM Phosphatebuffer, and the resultant antibody preparation then was added withmixing to the selenium colloid suspension prepared as describedhereinabove, to a final antibody concentration of 75 μg/ml. Stirring wascontinued for 40 minutes. Then, 1% (by weight) bovine serum albumin(BSA) was added to the solution, and the selenium colloid/antibodyconjugate solution was stirred for an additional 15 minutes andcentrifuged at 5000×g for 90 minutes. Following this, 90% of thesupernatant was removed, and the pellet was resuspended with theremaining supernatant. Immediately prior to coating this selenium-IgGconjugate to a glass fiber pad, it was diluted 1:10 with conjugatediluent (1% [by weight] casein, 0.1% [weight] Triton X-405®, and 50 mMTris, pH 8.2).

3. Procedural control reagent was prepared as a mixture of HIV-1 (groupM), HIV-1 (group O), and HIV-2 positive sera, and is utilized on aseparate strip device as a positive control of the assay.

4. Negative control reagent used was normal human utilized on a separatetest device as a negative control of the assay.

B. Application Pad Preparation.

The application pad material comprises resin bonded glass fiber paper(Lydall). Approximately 0.1 ml of the prepared conjugate (described inpreceding paragraph 2) is applied to the application pad.

C. Chromatographic Material Preparation.

All reagents are applied to a nitrocellulose membrane by charge anddeflect reagent jetting. The nitrocellulose is supported by a MYLAR®membrane that is coated with a pressure sensitive adhesive.

The test sample capture reagents were prepared by (a) diluting thespecific antigen prepared as described hereinabove to a concentration of0.5 mg/ml in jetting diluent (100 mM Tris, pH 7.6 with 1% sucrose (byweight), 0.9% NaCl and 5 μg/ml fluorescein) for HIV-1 group O capturereagent (pGO-9/CKS, SEQ ID NO:50), (b) for HIV-1 group M, subgroup Bcapture reagent (pTB319, SEQ ID NO:56), and (c) for HIV-2 capturereagent (pHIV-210, SEQ ID NO:55). 0.098 μl of a first capture reagent(reagent HIV-1 group M subgroup B; SEQ ID NO:56) was applied to thestrip at the designated capture location and constituted one patientcapture site. Likewise, 0.098 μl of a second capture reagent (reagentHIV-1 group O; SEQ ID NO:50) was applied to the strip at the designatedcapture location and constituted one patient capture site, and 0.098 μlof a third capture reagent (reagent HIV-2; SEQ ID NO:55) was applied tothe strip at the designated capture location and constituted one patientcapture site.

D. Rapid Assay for the Presence of Antibodies to HIV.

A rapid assay for the presence of antibodies to HIV in test samplesserum, whole blood, saliva, and urine samples was performed as follows.In a 1.5 ml Eppendorf tube, 5 μl of serum and 600 μl of sample elutionbuffer (SEB) (containing 50 mM Tris, 1% BSA (w/v), 0.4% Triton X-405®(v/v), 1.5% Casein (w/v), 3% Bovine IgG (w/v), 4% E. coli lysate (v/v),[pH 8.2]) was mixed. Four drops of this mixture was applied to thesample well of the STAR housing. Next, 1 μl of serum or whole blood wasadded to 100 μl of SEB in a well of a microtiter plate, and thenitrocellulose strip was added in the well. Following this, 1 μl ofserum or whole blood was spotted in the test device of the invention'ssample well directly and 4 drops of SEB was added. When testing saliva,50 or 75 μl of saliva was added to 50 μl or 25 μl of SEB, respectively,in a well of a microtiter plate, and the nitrocellulose test strip thenwas added to the well. When testing urine, 50 μl of urine was added to50 ul of SEB in a well of a microtiter plate, and the nitrocellulosetest strip was added in the well. Alternatively, 100 μl of urine wasused in the well of a microtiter plate, and the nitrocellulose teststrip was added, without using SEB.

The IgG in the sample was bound by the selenium-goat anti-human IgGcolloid in the conjugate pad, and the complexes were chromatographedalong the length of the nitrocellulose membrane test strips on which thethree recombinant antigens pGO-9 CKS SEQ ID NO:50), pTB319 (HIV-1 groupM (subgroup B), SEQ ID NO:56) and pHIV210 (HIV-2, SEQ ID NO:55)previously were applied at a concentration of 1 mg/ml using a biodotmachine, which provided positive displacement dispensing using precisedrop sizes. The test device then was incubated at room temperature fortwo minutes, and the results were read visually.

E. Spiked Whole Blood Assay.

In a 1.5 ml Eppendorf tube, the equivalent of 1 μl blood from eitherconfirmed positive HIV-1 group O, HIV-1 group M or HIV-2, or confirmednegative for HIV-1 group O, HIV-1 group M or HIV-2 whole blood testsample was added to 5 μl of a confirmed negative HIV-1 group O, HIV-1group M or HIV-2 serum along with 100 μl of SEB, and mixed. This mixturewas applied to the sample well of the test device of the invention.

The IgG in the sample was bound by the selenium-goat anti-human IgGcolloid in the conjugate pad, and the complexes were chromatographedalong the length of the nitrocellulose membrane test strips on which thethree recombinant antigens pGO-9 CKS SEQ ID NO:50), pTB319 (HIV-1 groupM (subgroup B), SEQ ID NO:56) and pHIV210 (HIV-2, SEQ ID NO:55)previously were applied at a concentration of 1 mg/ml using a biodotmachine, which provided positive displacement dispensing using precisedrop sizes. The test device then was incubated at room temperature fortwo minutes, and the results were read visually.

F. Results.

If antibody to antigen 1 was present in the test sample, a visiblereaction was indicated in the capture zone area of antigen 1 and in theassay completion zone, and not in the zones of antigen 2 or antigen 3.If antibody to antigen 2 was present in the test sample, a visiblereaction was indicated in the capture zone area of antigen 2 and in theassay completion zone, and not in the zones of antigen 1 or antigen 3.If antibody to antigen 3 was present in the test sample, a visiblereaction was indicated in the capture zone area of antigen 3 and in theassay completion zone, and not in the zones of antigen 1 or antigen 2.Also, a negative control should be non-reactive (show no visiblereaction) in the zones of antigen 1, antigen 2 and antigen 3, but shouldbe reactive in the assay completion zone. A positive control (knownreactive antibody to antigen 1, 2 and/or 3) should be reactive in thezone of the appropriate antigen to which it specifically binds in anantigen/antibody reaction. A result was considered invalid when apositive reaction occurred in one of the antigen capture zones but notin the assay completion zone, and the test was repeated.

(i) Assaying for Antibodies in Blood, Urine and Saliva. The blood,urine, and saliva of three patients (identified by patient numbers 0109,4068, and 4475) were tested on nitrocellulose solid phase devices of theinvention as described herein and following the assay protocol as setforth hereinabove. Each blood and urine test sample of each patient0109, 4068 and 4475 was reactive with antigen 1 (pTB319; SEQ ID NO:56).The saliva test sample of patients 4068 and 4475 also were reactive withantigen 1, while patient 0109's saliva test sample was non-reactive inthe test device of the invention. The saliva test sample of patient 0109was later retested by a standard EIA and confirmed non-reactive forantibodies to HIV-1 gp41, indicating that the results obtained for thesaliva test sample of patient 0109 were valid.

(ii) Assaying Negative Samples for HIV Antibodies. Two negative sera andtwo negative whole blood test samples, each spiked with the same twonegative sera, were tested. Samples contained no antibodies specific forthe relevant antigens and the test samples were negative after assay onthe test (i.e. no reactivity, as indicated by no visible bar signifyinga reaction in either position O, M or 2). Test sample was present ineach test device, as indicated by a positive reaction bar in the testsample reactivity zone.

(iii) Assaying for HIV-1 Group M Antibody. Five HIV-1 Group M sera andfive whole blood samples spiked with the HIV-1 Group M positive serawere tested using ten devices. HIV-1 Group M samples were seen tocontain antibodies specific for HIV-1 Group M antigen (pTB319) as shownby development of a reaction line at the HIV-1 Group M antigen zone, andvisible reaction lines could be seen in the assay completion zone ofnine out of 10 test devices. Although a band was present in oneparticular test device in the capture zone for HIV-1 group M antibody,test sample did not reach the assay completion zone and, thus, the assayneeded to be repeated for this particular sample. No cross-reactivitywas observed with the capture reagents for HIV group O and HIV-2.

(iv) Assaying for HIV-1 Group O Antibodies. Two confirmed positive HIV-1Group sera and two whole blood test samples spiked with HIV-1 Group Osera were tested using an additional four devices. The HIV-1 Group Osamples were found to contain antibodies specific for HIV-1 Group Oantigen as indicated by a positive bar result in the HIV-1 Group antigencapture zone area, with reaction lines visible in the assay completionzone of each device. No cross-reaction with HIV-1 group M or HIV-2capture antigens (no visible bar) was observed.

(v) Assaying for HIV-2 Antibodies. Ten further test devices were used totest five HIV-2 confirmed positive sera and whole blood spiked with the5 HIV-2 sera. The HIV-2 samples were found to contain antibodiesspecific for HIV-2 antigen (pHIV210) as shown by reaction bars at theHIV-2 antigen zone. No reaction was observed between these test samplesand the HIV-1 Group O or HIV-1 Group M antigens; visible reaction lineswere seen in the assay completion zone of each device.

(vi) Assaying HIV-1 Group M, HIV-1 Group O, HIV-2 and Negative Samples.Four final devices were used to test an HIV-1 Group M-positive testsample, an HIV-1 Group O-positive test sample, an HIV-2-positive testsample and a negative control sample. The negative test serum did notreact with any antigen in the antigen capture zone; the HIV-1 GroupM-positive test sample was reactive only with the HIV-1 Group M antigen;the HIV-1 Group O-positive test sample was reactive only with the HIV-1Group O antigen; and the HIV-2-positive test sample was reactive onlywith the HIV-2 antigen. Visible reaction lines were seen in the assaycompletion zone of each device.

The five HIV-1 group M and the two HIV-1 group O test samples used wereconfirmed seropositive samples which had been previously tested using acommercially-available enzyme immunoassay (Abbott #3A77) and had beenPCR amplified, sequenced and subtyped based on phylogenetic analysis.The five HIV-2 samples used were seropositive using the same EIA andwere confirmed as HIV-2-positive samples using an HIV-2 Western blottest (Sanofi).

EXAMPLE 11 Construction of Synthetic HIV-1 Group M and HIV-1 Group OHybrid Genes

A. Modification of pTB319

The plasmid pTB319 (U.S. Pat. No. 5,124,255, incorporated herein byreference) encodes a truncated gp41 recombinant protein due to a onebase deletion within the synthetic HIV-1 Group M gp41 gene resulting ina frame-shift. In order to facilitate the generation of HIV-1 Group Mand Group O hybrid gene constructs, site-specific mutagenesis was usedto eliminate the frame-shift within the gp41 coding region in pTB319.This was accomplished by sequentially digesting the plasmid pTB319 withthe restriction endonucleases Rsr II and Bst XI. The syntheticoligonucleotides pTB319+A (SEQ ID NO:98) and pTB319+T (SEQ ID NO:99)were annealed and ligated into the Rsr II and Bst XI digested pTB319.The ligation product was used to transform supercompetent XL1-Blue cellsand the cells were plated on LB agar plates supplemented with 150 μg/mlampicillin. Colony PCR was used to identify correctly modified clonesusing the primer combinations pTB-S4 (SEQ ID NO:100)/pTB-S7 (SEQ IDNO:101) and pTB-S4 (SEQUENCE ID NO:100)/63168 (SEQ ID NO:121). Overnightcultures were established for candidate clones in LB broth supplementedwith 3 mM glucose and 200 μg/ml ampicillin for preparation of miniprepDNA. The entire coding region was sequenced using the oligonucleotideprimers: δ3461 (SEQ ID NO:2), 43285 (SEQ ID NO:1), CKS-1 (SEQ ID NO:30),CKS-3 (SEQ ID NO:32), pTB-S1 (SEQ ID NO:102), pTB-S2 (SEQ ID NO:103),pTB-S3 (SEQ ID NO:104), pTB-S4 (SEQ ID NO:100), pTB-S5 (SEQ ID NO:105),pTB-S6 (SEQ ID NO:106), pTB-S7 (SEQ ID NO:101), and pTB-S8 (SEQ IDNO:28). Based on sequencing results, clone pTB319+A-#31 (pGMcks-1) hasthe desired coding region sequence. This clone was subsequentlydesignated as pGM-1CKS/XL1 (SEQ ID NO:107 presents the nucleotidesequence of the coding region). SEQ ID NO:108 is the amino acid sequenceof the pGM-1CKS recombinant protein.

B. Construction of pGO-12CKS/XL1

The construct pGO-12CKS/XL1 encodes the recombinant protein pGO-12CKS,the amino acid sequence of which is shown in SEQ ID NO:91. This proteinconsists of 250 amino acids of CKS/polylinker fused to 42 amino acids ofenv gp120 (HIV-1 Group M, HXB2R isolate), 200 amino acids of env gp41(HIV-1 Group M, HXB2R isolate), 45 amino acids of env gp120 (HIV-1 GroupO, HAM112 isolate), and 199 amino acids of env gp41 (HIV-1 Group O,HAM112 isolate). pGO-12CKS/XL1 was constructed as follows:

A PCR reaction (100 μl volume) was set up with UlTma DNA Polymerase (3U) and 1× buffer along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 μmol ofpTB/O-5′ (SEQ ID NO:109), 50 μmol pGO-9/Kpn (SEQ ID NO:110), and 1 ngpGO-9PL DNA (miniprep H5; obtained from Example 3, Section F above) astemplate. The reaction was incubated at 94° C. for 105 seconds thenamplified with 22 cycles of 94° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 75 seconds, followed by incubation at 72° C. for5 minutes. The pTB/O-5′/pGO-9/Kpn PCR product was isolated on gel. ThepTB/O-5′/pGO-9/Kpn PCR product and pGM-1CKS plasmid (described inSection A hereinabove) were digested sequentially with Asp 718(Boehringer Mannheim Biochemicals) and Bst XI. The digested vector wasthen treated with calf intestinal alkaline phosphatase (BoehringerMannheim Biochemicals), extracted with phenol/chloroform, andprecipitated with ethanol. The digested PCR product was purified on aCentri-Sep column (Princeton Separations). Digested PCR product wasligated into the digested and phosphatased pGM-1CKS vector overnight at16° C. XL1-Blue supercompetent cells were transformed with the ligationproduct and plated on LB+ampicillin plates supplemented with 20 mMglucose. Colonies were restreaked for isolation on the same type ofplates. An overnight culture (LB medium+100 μg/ml carbenicillin+20 mMglucose) of clone pGO-12CKS clone #1 was set up. Frozen stocks (0.5 ml80% glycerol+0.5 ml overnight culture) were made and miniprep DNA wasprepared for sequencing. The following oligonucleotides were used asprimers for sequence analysis: CKS-1 (SEQ ID NO:30), CKS-2 (SEQ IDNO:31), CKS-3 (SEQ ID NO:32), CKS-4 (SEQ ID NO:33), CKS 176.1 (SEQ IDNO:19), 3962 (SEQ ID NO:111), 3965 (SEQ ID NO:113), pTB-S2 (SEQ IDNO:103), pTB-S3 (SEQ ID NO:104), pTB-S4 (SEQ ID NO:100), pTB-S5 (SEQ IDNO:105), sy120-S1 (SEQ ID NO:112), 41sy-1B (SEQ ID NO:29), 41sy-2B (SEQID NO:34), 41sy-4 (SEQ ID NO:23), pTB-S8 (SEQ ID NO:28). Based on theresults of the sequence analysis, pGO-12CKS candidate clone #1 wasdesignated as pGO-12CKS/XL1. (SEQ ID NO:90 presents the nucleotidesequence of the coding region, and SEQ ID NO:91 presents the encodedamino acid sequence.)

C. Construction of pGO-13CKS/XL1

The construct pGO-13CKS/XL1 encodes the recombinant protein pGO-13CKS,the amino acid sequence of which is shown in SEQ ID NO:93. This proteinconsists of 250 amino acids of CKS/polylinker fused to 42 amino acids ofenv gp120 (HIV-1 Group M, HXB2R isolate), 200 amino acids of env gp41(HIV-1 Group M, HXB2R isolate), 45 amino acids of env gp120 (HIV-1 GroupO, HAM112 isolate), and 169 amino acids of env gp41 (HIV-1 Group O,HAM112 isolate). pGO-13CKS/XL1 was constructed as follows:

A PCR reaction (100 μl volume) was set up with UlTma DNA Polymerase (3U) and 1× buffer along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 μmol ofpTB/O-5′ (SEQ ID NO:109), 50 μmol pGO-8/Kpn (SEQ ID NO:114), and 1 ngpGO-9PL DNA (miniprep H5; obtained from Example 3, Section Fhereinabove) as template. The reaction was incubated at 94° C. for 105seconds then amplified with 22 cycles of 94° C. for 30 seconds, 55° C.for 30 seconds, and 72° C. for 75 seconds, followed by incubation at 72°C. for 5 minutes. The pTB/O-5′/pGO-8/Kpn PCR product was isolated ongel. The pTB/O-5′/pGO-8/Kpn PCR product and pGM-1CKS plasmid (describedin Section A above) were digested sequentially with Asp 718 (BoehringerMannheim Biochemicals) and Bst XI. The digested vector was then treatedwith calf intestinal alkaline phosphatase (Boehringer MannheimBiochemicals), extracted with phenol/chloroform, and precipitated withethanol. The digested PCR product was purified on a Centri-Sep column(Princeton Separations). Digested PCR product was ligated into thedigested and phosphatased pGM-1CKS vector overnight at 16° C. XL1-Bluesupercompetent cells were transformed with the ligation product andplated on LB+ampicillin plates supplemented with 20 mM glucose. Colonieswere restreaked for isolation on the same type of plates. An overnightculture (LB medium+100 μg/ml carbenicillin+20 mM glucose) of clonepGO-13CKS clone #1 was set up. Frozen stocks (0.5 ml 80% glycerol+0.5 mlovernight culture) were made and miniprep DNA was prepared forsequencing. The following oligonucleotides were used as primers forsequence analysis: CKS-1 (SEQ ID NO:30), CKS-2 (SEQ ID NO:31), CKS-3(SEQ ID NO:32), CKS-4 (SEQ ID NO:33), 43461 (SEQ ID NO:2), 43285 (SEQ IDNO:1), pTB-S1 (SEQ ID NO:102), pTB-S2 (SEQ ID NO:103), pTB-S3 (SEQ IDNO:104), pTB-S4 (SEQ ID NO:100), pTB-S5 (SEQ ID NO:105), sy120-S1 (SEQID NO:112), 41sy-1B (SEQ ID NO:29), 41sy-2B (SEQ ID NO:34), 41sy-4 (SEQID NO:23), pTB-S8 (SEQ ID NO:28). Based on the results of the sequenceanalysis, pGO-13CKS candidate clone #1 was designated as pGO-13CKS/XL1.(SEQ ID NO:92 presents the nucleotide sequence of the coding region, andSEQ ID NO:93 presents the encoded amino acid sequence.)

D. Construction of pGO-14PL/DH5α

The construct pGO-14PL/DH5 (encodes the recombinant protein pGO-14PL,the amino acid sequence of which is shown in SEQ ID NO:95. This proteinconsists of an N-terminal methionine followed by 45 amino acids of envgp120 (HIV-1 Group O, HAM112 isolate), 200 amino acids of env gp41(HIV-1 Group O, HAM112 isolate) fused to 42 amino acids of env gp120(HIV-1 Group M, HXB2R isolate), and 200 amino acids of env gp41 (HIV-1Group M, HXB2R isolate). pGO-14PL/DH5α was constructed as follows:

A PCR reaction (100 μl volume) was set up with UlTma DNA Polymerase (3U) and 1× buffer along with 1.5 mM MgCl₂, 40 μM of each dNTP, 50 μmol ofpTB/Age5′ (SEQ ID NO: 115), 50 μmol pGO/B-3′ (SEQ ID NO:116), and 1 ngpGM-1CKS DNA (miniprep of pTB319+A-#31; obtained from Section A above)as template. The reaction was incubated at 95° C. for 30 seconds thenamplified with 22 cycles of 94° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 60 seconds, followed by incubation at 72° C. for5 minutes. The pTB/Age5′/pGO/B-3′ PCR product was isolated on gel. ThepTB/Age5′/pGO/B-3′ PCR product and pGO-9PL plasmid (obtained fromExample 3, Section F hereinabove) were digested sequentially with Age Iand Bam HI. The digested vector was then treated with calf intestinalalkaline phosphatase (Boehringer Mannheim Biochemicals), extracted withphenol/chloroform, and precipitated with ethanol. The digested PCRproduct was purified on a Centri-Sep column (Princeton Separations).Digested PCR product was ligated into the digested and phosphatasedpGM-1CKS vector overnight at 16° C. DH5α competent cells weretransformed with the ligation product and plated on LB+ampicillin (150μg/ml) plates. Colonies were analyzed for the presence of the properinsert by colony PCR using the vector primers pKRR EcoR1 forward (SEQ IDNO:38) and pKRR BamH1 reverse (SEQ ID NO:39). Colonies containingcandidate clones were restreaked for isolation on the same type ofplates. Overnight cultures (LB medium+100 μg/ml carbenicillin) were setup to generate frozen stocks and miniprep DNA. Frozen stocks (0.5 ml 80%glycerol+0.5 ml overnight culture) were made and miniprep DNA wasprepared for sequencing. The following oligonucleotides were used asprimers for sequence analysis: pTB-S1 (SEQ ID NO:102), pTB-S2 (SEQ IDNO:103), pTB-S3 (SEQ ID NO:104), pTB-S4 (SEQ ID NO:100), pTB-S5 (SEQ IDNO:105), 41sy-1C (SEQ ID NO:40), 41sy-2 (SEQ ID NO:41), 41sy-3 (SEQ IDNO:42), 41sy-4 (SEQ ID NO:23), pKRREcoR1 forward (SEQ ID NO:38), pKRRBamH1 reverse (SEQ ID NO:39). Based on the results of the sequenceanalysis, pGO-14PL candidate clone #11 was designated as pGO-14PL/DH50α.(SEQ ID NO:94 presents the nucleotide sequence of the coding region, andSEQ ID NO:95 presents the encoded amino acid sequence.)

EXAMPLE 12 Construction of a HIV-1 Group O env gp120/gp41 Synthetic Genewith a Second Copy of the gp41 Immunodominant Region (IDR) Fused to theC-terminus

A. Construction of pGO-15CKS/XL1

The construct pGO-15CKS/XL1 encodes the recombinant protein pGO-15CKS,the amino acid sequence of which is shown in SEQ ID NO:97. This proteinconsists of 246 amino acids of CKS/polylinker fused to 45 amino acids ofenv gp120 (HIV-1 Group O, HAM112 isolate), 199 amino acids of env gp41(HIV-1 Group O, HAM112 isolate), followed by a 4 amino acid linker (Gly,Gly, Gly, Ser) and 32 amino acids encompassing the IDR region of envgp41 (HIV-1 Group O, HAM112 isolate). pGO-15CKS/XL1 was constructed asfollows:

The plasmid pGO-11CKS propagated in XL1-Blue cells (obtained fromExample 3, Section K) was digested sequentially with Age I and Bam HI,extracted with phenol/chloroform, and precipitated with ethanol. Thesynthetic oligonucleotides synIDR#2-A (SEQ ID NO:117) and synIDR#2-B(SEQ ID NO:118) were kinased with polynucleotide kinase (BoehringerMannheim Biochemicals) following the manufacturer's recommendedprocedure. The kinased oligonucleotides were annealed and the duplexligated to the digested (Age I+Bam HI) pGO-11CKS vector. SupercompetentXL1-Blue cells were transformed with the ligation product, and the cellswere plated on LB plates supplemented with 150 μg/ml ampicillin andincubated overnight. Colony PCR (primers 41sy-1B SEQ ID NO:29 and pTB-S8SEQ ID NO:28) was used to identify candidate clones. Colonies wererestreaked for isolation on LB plates supplemented with 150 μg/mlampicillin. Overnight cultures of the candidate clones were establishedin 2×LB broth (Life Technologies, Inc.) supplemented with 100 mg/mlcarbenicillin and 20 mM glucose (Sigma Chemical Co.). Miniprep DNA wasprepared from the overnight cultures using a Promega 373 DNA isolationkit (Promega Corporation, Madison, Wis.) following the manufacturer'srecommended procedure. The overnight cultures were also used toestablish frozen stocks. Cells were pelleted and resuspended in 2×LBbroth with 20% glycerol (J. T. Baker, Phillipsburg, N.J.) and frozen at−70° C. The following oligonucleotide primers were used for sequenceanalysis: CKS-1 (SEQ ID NO:30), CKS-3 (SEQ ID NO:32), 43285 (SEQ IDNO:1), 43461 (SEQ ID NO:2), 41sy-1B (SEQ ID NO:29), 41sy-2B (SEQ IDNO:34), 41sy-3B (SEQ ID NO:35), 41sy-4 (SEQ ID NO:23), and CKS3583 (SEQID NO:20). Based on sequencing results, candidate clone pGO-15CKS-48 wasdesignated as pGO-15CKS/XL1. (SEQ ID NO:96 presents the nucleotidesequence of the coding region, and SEQ ID NO:97 presents the encodedamino acid sequence.)

B. Construction of pGO-15PL/DH50α.

The construct pGO-15PL/DH50α encodes the recombinant protein pGO-15PL,the amino acid sequence of which is shown in SEQ ID NO:120. This proteinconsists of an N-terminal methionine followed by 45 amino acids of envgp120 (HIV-1 Group O, HAM112 isolate), 199 amino acids of env gp41(HIV-1 Group O, HAM112 isolate), a 4 amino acid linker (Gly, Gly, Gly,Ser) and 32 amino acids encompassing the IDR region of env gp41 (HIV-1Group O, HAM112 isolate). pGO-15PL/DH5 was constructed as follows:

A PCR reaction (100 μl volume) was set up with AmpliTaq DNA Polymerase(2.5 U) and 1× buffer along with 40 μM of each dNTP, 50 μmol of 41sy-3B(SEQ ID NO:35), 50 μmol pTB-S8 (SEQ ID NO:28), and 1 ng pGO-15CKS DNA(miniprep of candidate clone pGO-15CKS-48; obtained from Section Aabove) as template. The reaction was incubated at 95° C. for 30 seconds,then amplified with 35 cycles of 94° C. for 20 seconds, 50° C. for 30seconds, and 72° C. for 60 seconds, followed by incubation at 72° C. for7 minutes. The amplified product was purified using a QIAquick PCRPurification Kit (Qiagen). The purified 41sy-3B/pTB-S8 amplificationproduct was digested sequentially with Age I and Bam HI, then ligated topGO-9PL (Age I+Bam HI digested/phosphatased vector prep from Example 3,Section J above). Competent DH5α cells were transformed using theligation product and plated on LB plates supplemented with 150 μg/mlampicillin. Candidate clones were identified by colony PCR with theprimers 41sy-3 (SEQ ID NO:42) and pKRR BamHI reverse (SEQ ID NO:39),followed by digestion of the PCR product with Age I. Candidate clone #4was restreaked for isolation. A culture of clone #4 was established in2×LB broth (Life Technologies) supplemented with 100 μg/ml carbenicillin(Sigma Chemical Co.) and incubated at 34° C. overnight. Miniprep DNA wasprepared from part of the overnight culture using a Promega 373 DNAIsolation Kit (Promega Corp.) as outlined by the manufacturer. Frozenstocks were established by pelleting the remaining overnight culture andresuspending the cells in Terrific Broth with 20% glycerol (J. T. BakerCo.) and freezing at −70° C. The following oligonucleotide primers wereused for sequence analysis: pKRR EcoR1 forward (SEQ ID NO:38), pKRRBamHI reverse (SEQ ID NO:39), 41sy-1C (SEQ ID NO:40), 41sy-2 (SEQ IDNO:41), 41sy-3 (SEQ ID NO:42), 41sy-3B (SEQ ID NO:35) and 41sy-4 (SEQ IDNO:23). Based on sequencing results, candidate pGO-15PL clone #4 wasdesignated as pGO-15PL/DH5α. (SEQ ID NO:119 presents the nucleotidesequence of the coding region, and SEQ ID NO:120 presents the encodedamino acid sequence.)

EXAMPLE 13 Preparation and Purification of HIV-1 Group O Recombinantgp41 Antigens pGO-8 PL, pGO-9 PL, pGO-12CKS, pGO-14 PL and pGO-15CKS

The above antigens were prepared by growing and inducing E. coli strainscontaining the respective HIV-1 Group O recombinant gp41 antigenconstructs as described in Example 5. The resulting frozen cells wereresuspended by homogenization in cold lysis buffer comprising 50 mM TrispH 8, 10 mM Na EDTA, 150 mM NaCl, 8% (w/v) sucrose, 5% Triton X-100®(v/v), 1 mM PMSF and 1 mM pepstatin A. Lysozyme was added to thehomogenates at a concentration of 1.3 mg per gram of cells processed,and incubated for 30 minutes on ice to lyse the cells. Inclusion bodieswere separated from soluble proteins by centrifugation. These pelletedinclusion bodies were washed and pelleted sequentially in 1) LysisBuffer; 2) 10 mM Na EDTA pH 8, 30% (w/v) sucrose; and 3) water. Thewashed inclusion bodies were resuspended in 50 mM Tris pH 8, 10 mM NaEDTA, 150 mM NaCl and 3 M urea, and incubated on ice for 1 hour. Theinclusion bodies then were separated from the solubilized proteins bycentrifugation. The pelleted inclusion bodies were fully solubilized in7 M guanidine-HCl, 50 mM Tris pH 8, 0.1% (v/v) beta-mercaptoethanol(BME) overnight at 4° C. The solubilized recombinant antigen(s) wereclarified by centrifugation, passed through a 0.2 μm filter. Thesolubilized gp41 antigen(s) were precipitated from the 7 M Guanidine-HClsolution by dilution (1:7) with water to a final concentration of 1 MGuanidine-HCl. After incubation at 4° C. for 30 minutes, theprecipitated proteins were centrifuged and resolubilized in 50 mM TrispH 8, 9 M Urea, 0.1% BME (v/v) overnight at 4° C.

Solubilized HIV-1 Group O recombinant gp41 antigens were next purifiedas follows: The recombinant antigens were first purified by anion and/orcation exchange chromatography using Q-Sepharose (Pharmacia) orS-Sepharose (Pharmacia) columns. The solubilized gp41 antigen solutionswere loaded onto either a Q-Sepharose or S-Sepharose column that hadbeen previously equilibrated with 50 mM Tris pH 8, 8M Urea, 0.1% BME(v/v). The gp41 antigens either (1) passed though the column directlyand were collected in the void volume or (2) were bound to the columnmatrix. If adsorbed, the gp41 antigens were eluted from the columns by a0-1M NaCl gradient. SDS-polyacrylamide gel electrophoresis was used toanalyze fractions from the Q-Sepharose or S-Sepharose columns. Fractionscontaining the recombinant gp41 antigens were pooled and thenconcentrated by ultrafiltration. The recombinant antigen concentrateswere treated with 4% SDS (w/v) and 5% BME (w/v) at room temperature forthree hours. SDS treated antigens were further purified by sizeexclusion chromatography on a Sephacryl S-300 (Pharmacia) columnequilibrated with 25 mM Tris pH 8, 0.15 M NaCl, 0.1% v/v BME, 0.1% SDS(w/v). SDS-polyacrylamide gel electrophoresis was used to analyze thefractions from the S-300 column. Fractions containing purifiedrecombinant antigens were pooled, passed through a 0.2 μm filter andstored at −70° C.

EXAMPLE 14 Test of Recombinant Antigen Reactivity with HIV-1 Group M andGroup O Samples

A. Bead Coating

In order to examine the reactivity of recombinant HIV-1 antigens,purified recombinants were coated on quarter inch polystyrene beads.These antigen coated beads were used in a series of capture assays toaccess reactivity to both HIV-1 Group M and Group O samples.

Recombinant antigens were coated on quarter inch beads at 0.5 μg/ml inPBS. The following recombinant antigens were coated: pTB319 (Group M),pGO-9/CKS, pGO-15/PL, pGO-12/CKS, pGO-14/PL and pGO-15/CKS (all GroupO).

The procedure for coating the recombinant antigens on the beads is asfollows: For each antigen, 35.5 gm. (˜250) of beads, (AbbottLaboratories code 93-2556, lot 6840M100), were washed in 15% N-propanolin water for 30 minutes at 40° C. All incubations and washes were donein small brown glass jars on a shaker platform. The N-propanol solutionwas aspirated off, 58.25 ml of antigen solution was added, and the beadswere incubated for two hours at 40° C. The antigen solution wasaspirated off, and 60 ml of a 0.1% Triton X-100 solution in PBS wasadded for 30 minutes at 40° C. The beads were then washed with 60 ml ofPBS two times and incubated with 60 ml of 2% BSA in PBS for 30 minutesat 40° C. The BSA was aspirated and the beads were washed again in PBS.The beads were then incubated with 60 ml 0.5% sucrose in PBS for 15minutes at room temperature. After 15 minutes, the sucrose was aspiratedand the beads were allowed to air dry. Coated beads were stored inpolypropylene bottles with a desiccant at 4° C.

B. Assays

Recombinant antigen coated beads were tested for reactivity against avariety of samples using the Abbott Laboratories 3A11 kit (firstgeneration, indirect assay format). Samples were diluted and added towells in polystyrene trays. Beads were added and the trays wereincubated at 40° C. for 1 hour. The trays were washed with water in anAbbott Laboratories QUICKWASH device. Next the kit conjugate, ananti-human IgG-Horseraddish Peroxidase, was added and the trays wereagain incubated at 40° C. for one hour. The trays were again washed and300 μl of substrate solution, (1.28 mg/ml o-Phenylenediamine•HCl inCitrate-Phosphate buffer containing 0.02% Hydrogen Peroxide), was addedto each well for 30 minutes at room temperature. 1 ml of 1N sulfuricacid was added to stop the reaction, and the trays were read in anAbbott QUANTUM spectrophotometer.

The samples used for this study were Normal human plasma, (AbbottLaboratories code 99800, lot 17535M400), used as a negative control;HIVPL-31 (Group M positive sera), and the following Group O positivesera: 14283, 189404, 193Ha, 14791, 267Ha and ESP-1. All samples exceptthe Normal human plasma control were run at three dilutions; 1:1,000,1:10,000 and 1:100,000 in the kit specimen diluent. Each dilution ofeach sample was run in duplicate against each of the six beads, and theresults of each dilution were averaged and plotted for each bead.

C. Results

The results of the above tests demonstrate the improvements insensitivity and selectivity available by use of the recombinant antigensof the present invention. The bead coated with the HIV-1 Group Mrecombinant antigen (pTB319) detected the Group M serum sample, butfailed to detect all but one of the Group O samples. The beads coatedwith only HIV-1 Group O recombinant antigens (pGO-9/CKS, pGO-11/PL, andPGO-15/CKS) detected the Group O serum samples, but showed lowersensitivity in detection of the HIV-1 Group M sample. Beads that werecoated with hybrid Group M and Group O recombinant antigens (pGO-12/CKS,and pGO-14/PL) were able to detect both HIV-1 Group M- and GroupO-positive samples. Lastly, pGO-15/CKS, which has an additional sequencerepresenting the Group O immunodominant region of gp41 linked byrecombinant means to the carboxy end of the protein, showed greaterreactivity to low-titer Group O samples.

EXAMPLE 15 Examination of Assay Sensitivity for HIV-1 Group O-InfectedSamples Using Group O Recombinant Antigens pGO-9CKS and pGO-11CKS

A. Assays

In order to evaluate the performance in immunoassays of antigenconstructs of the present invention, recombinant antigens pGO-9CKS andpGO-11CKS were incorporated into four HIV-1/HIV-2 immunoassayscontaining HIV-1 Group M (subtype B) reagents. The constructs weretested using one bead assay (Assay 1) and 3 automatedmicroparticle-based assays (Assays 2-4). In all cases, the reactivity ofHIV-1 Group O-infected specimens was assessed with (format 2) andwithout (format 1) incorporation of the HIV-1 group O recombinants. Thecoated beads/microparticles were reacted with multiple dilutions of thefollowing HIV-1 Group O-positive human sera: ESP1, 189404, 193Ha, 341Ha, 2156 and ABB 9/96.

For Assay 1, purified pGO-11CKS was incorporated into acommercially-available bead-based assay by coating the antigen constructonto quarter-inch polystyrene beads. The coated beads were reacted witha range of dilutions of HIV-1 Group O-positive human sera, washed, andthen reacted with purified pGO-9CKS conjugated to horseradishperoxidase. After washing/separation of bound from unbound pGO-9CKSconjugate, substrate was added and the assay was completed as indicatedin Example 14.

For Assay 2, purified pGO-11CKS was incorporated into a secondcommercially-available assay by coating the antigen construct ontomicroparticles. The coated microparticles were reacted with the samerange of dilutions of HIV-1 Group O-positive human sera utilized inAssay 1. The microparticles were then washed and subsequently reactedwith biotinylated pGO-9CKS. After further washing, the microparticleswere reacted with a polyclonal anti-biotin antibody conjugated toalkaline phosphatase. The assay signal was developed by addition of thesubstrate methylumbelliferyl phosphate.

For Assay 3, purified pGO-11CKS was incorporated into a thirdcommercially-available assay by coating the antigen construct onmicroparticles. The coated microparticles were again reacted with thesame range of dilutions of HIV-1 Group O-positive human sera utilized inAssay 1. Next, the microparticles were washed and then reacted withbiotinylated pGO-9CKS. After washing, the microparticles were reactedwith an anti-biotin antibody conjugated to acridinium as thesignal-generating compound.

For Assay 4, purified pGO-11CKS was incorporated into a developmentalassay by coating the antigen construct onto magnetic microparticles. Asin Assay 1, the coated microparticles were reacted with a range ofdilutions of HIV-1 Group O-positive human sera, washed, and subsequentlyreacted with pGO-9CKS conjugated to acridinium.

B. Results

The results of the above tests are presented as signal/cutoff (S/CO)ratios. Format 1 refers to the conventional assay without the antigenconstructs of the present invention, while Format 2 refers to the assaysupplemented with the HIV-1 group O constructs.

From these data, it can be seen that the addition of the HIV-1 Group Orecombinants resulted in a significant enhancement of assay sensitivityfor the HIV-1 Group O-infected sera at all of the dilutions tested. Forexample, in the case of Assay 1 and sample 193Ha a S/CO ratio of 7.14was obtained at a 1:10 dilution using Format 1, while a similar S/CO(7.22) was obtained at a 160-fold greater dilution (1:1600) using Format2. This trend was maintained across all of the tested assay platforms.The utility of the group O recombinants was particularly evident forsample 2156, which tested negative (S/CO<1) in all 4 assays prior to theaddition of the group O recombinants. With the addition of the HIV-1Group O constructs, however, this sample 2156 tested positive in allfour assays at a 1:400 dilution. In Assay 1, 2156 was still positive ata dilution of 1:5000. Addition of the recombinant reagents pGO-9CKS andpGO-11CKS was thus seen to provide a substantially better sensitivityfor HIV-1 Group O-infected sera when using the above direct-formatimmunoassays.

1. An isolated antigen construct wherein said antigen constructcomprises a fusion protein, said fusion protein comprising a first HIV-1Group O gp120 polypeptide fused to a second HIV-1 Group O gp41polypeptide, further wherein the first polypeptide comprises amino acidresidues 476 through 520 of SEQ ID NO:61 and the second polypeptidecomprises amino acid residues 292 through 490 of SEQ ID NO:50.
 2. Anisolated antigen construct wherein said antigen construct comprises afusion protein, said fusion protein comprising a first HIV-1 Group Ogp120 polypeptide comprising amino acid residues 476 through 520 of SEQID NO:61 fused to a second HIV-1 Group O gp41 polypeptide wherein theantigen construct is pGO-9CKS.
 3. The isolated antigen construct ofclaim 2, wherein the construct comprises the amino acid sequence of SEQID NO:50.